EP1294771B1 - Chimeric GapC protein from Streptococcus and its use in vaccination and diagnosis - Google Patents

Chimeric GapC protein from Streptococcus and its use in vaccination and diagnosis Download PDF

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EP1294771B1
EP1294771B1 EP01944803A EP01944803A EP1294771B1 EP 1294771 B1 EP1294771 B1 EP 1294771B1 EP 01944803 A EP01944803 A EP 01944803A EP 01944803 A EP01944803 A EP 01944803A EP 1294771 B1 EP1294771 B1 EP 1294771B1
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sequence
gapc
protein
fusion polypeptide
multiple epitope
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German (de)
French (fr)
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EP1294771A2 (en
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Andrew A. Potter
Jose Perez-Casal
Michael Fontaine
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University of Saskatchewan
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University of Saskatchewan
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates generally to bacterial antigens and genes encoding the same. More particularly, the present invention pertains to the construction of a chimeric plasmin binding protein gene.
  • Mastitis an infection of the mammary gland usually caused by bacteria or fungus, results in major economic losses to the dairy industry yearly.
  • bacterial species most commonly associated with mastitis are various species of the genus Streptococcus , including S. aureus, S. uberis , (untypeable), S. agalactiae (Lancefield group B), S. dysgalactiae (Lancefleld group C), S. zooepidemicus , and the Lancefield groups D, G., L and N streptococci.
  • Some of those species are contagious (e.g. S. agalactiae ), while others are considered environmental pathogens (e.g. S.
  • S. uberis mastitis may also take the form of an acute clinical condition, with obvious signs of disease such as clots or discoloration of the milk and swelling or hardness of the mammary gland. Some cases of the clinical disease can be severe and pyrexia may be present.
  • ubenis mastitis see, Bramley (1991) Mastitis: physiology or pathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and A. W. Hill (ed.), New insights into the pathogenesis of mastitis. Rijksuniversiteit Gent, Belgium ; and Schalm et al. (1971) The mastitis complex-A brief summary. p. 1-3. In Bovine Mastitis. Lea & Febiger, Philadelphia
  • the literature is limited regarding vaccination studies with S. dysgalactiae and S. uberis , and variable results have been observed. In some cases, immunization has resulted in increased sensitivity to the specific organism and in other cases strain-specific protection has been obtained.
  • S. dysgalactiae is known to bind several extracellular and plasma-derived proteins such as fibronectin, fibrinogen, collagen, alpha-II-macroglobulin, IgG, albumin and other compounds.
  • the organism also produces hyaluronidase and fibrinolysin and is capable of adhering to and invading bovine mammary epithelial cells.
  • hyaluronidase and fibrinolysin is capable of adhering to and invading bovine mammary epithelial cells.
  • the exact roles of the bacterial components responsible for these phenotypes in pathogenesis is not known.
  • CAMP factor also known as UBERIS factor ( Skalka, B. and Smola, J. (1981) Monbl. Bakteriol. Ser. A 249:190-194 ), R-like protein, plasminogen activator and CAMP factor.
  • UBERIS factor Skalka, B. and Smola, J. (1981) Monbl. Bakteriol. Ser. A 249:190-194
  • R-like protein R-like protein
  • plasminogen activator plasminogen activator
  • the ⁇ antigen of the group B Streptococci strain A909 is a component of the c protein marker complex, which additionally comprises an ⁇ and ⁇ subunit ( Boyle, U.S. Patent No. 5,721,339 ). Subsets of serotype Ia, II, and virtually all serotype Ib cells of group B streptococci, have been reported to express components of the c protein. Use of the ⁇ subunit as an immunogenic agent against infections by Lancefield Group B Streptococcus infection has been proposed. However, its use to prevent or treat bacterial infections in animals, including mastitis in cattle, has not been studied.
  • GapC plasmin binding protein from a strain of Group A Streptococcus has previously been identified and characterized, and its use in thrombolytic therapies has been described ( Boyle, et al., U.S. Patent No. 5,237,050 ; Boyle, et al., U.S. Patent No. 5,328,996 ).
  • Boyle, et al., U.S. Patent No. 5,237,050 Boyle, et al., U.S. Patent No. 5,328,996
  • the use of GapC as an immieuxic agent to treat or prevent mastitis was neither described nor suggested.
  • the group A streptococcal M protein is considered to be one of the major virulence factors of this organism by virtue of its ability to impede attack by human phagocytes ( Lancefield, R.C. (1962) J. Immunol. 89:307-313 ).
  • the bacteria persist in the infected tissue until antibodies are produced against the M molecule.
  • Type-specific antibodies to the M protein are able to reverse the antiphagocytic effect of the molecule and allow efficient clearance of the invading organism.
  • M proteins are one of the key virulence factors of Streptococcus pyogenes , due to their involvement in mediating resistance to phagocytosis ( Kehoe, M.A. (1991) Vaccine 9:797-806 ) and their ability to induce potentially harmful host immune responses via their superantigenicity and their capacity to induce host-cross-reactive antibody responses ( Bisno, A.L. (1991) New Engl. J. Med. 325:783-793 ; Froude et al. (1989) Curr. Top. Microbiol. Immunol. 145:5-26 ; Stollerman, G.H. (1991) Clin. Immunol. Immunopathol. 61:131-142 ).
  • M proteins opsonic epitopes are extremely type-specific, resulting in narrow, type-specific protection.
  • some M proteins appear to contain epitopes that cross react with tissues of the immunized subject, causing a harmful autoimmune response (See e.g., Dale, J.L. and Beached, G.H. (1982) J. Exp. Med 156:1165-1176 ; Dale, J.L. and Beached, G.H. (1985) J. Exp. Med. 161:113-122 ; Baird, R.W., Bronze, M.S., Drabs, W., Hill, H.R., Veasey, L.G.
  • An octavalent M protein vaccine has been constructed and was tested for protective immunogenicity against multiple serotypes of group A streptococci infection in rabbits.
  • the immune response obtained was serotype-specific, conferring protection only against those bacterial strains exhibiting the M protein epitopes present in the chimeric protein ( Dale, J.B., Simmons, M. , Chiang, E.C., and Chiang, E.Y. (1996) Vaccine 14:944-948 ).
  • Chimeric proteins containing three different fibronectin binding domains derived from fibronectin binding proteins of S. dysgalactiae and Staphylococcus aureus have been expressed on the surface of Staph. carnosus cells.
  • FNBDs fibronectin binding domains
  • intranasal immunizations with live recombinant Staph. carnosus cells expressing the chlineric protein on their surface resulted in an improved antibody response to a model immunogen present within the chimeric surface protein.
  • a chimeric Protein G molecule (a type III Fc binding protein specific for the Fc region of all subclasses of IgG antibody molecules) is known, but its use as an immunogenic agent has not been described or suggested ( Bjorck, et al. (1992) U.S. Patent No. 5,108,894 ).
  • WO 98/18930 describes antigenic peptides from Steptococcus pneumoniae , including a peptide which may correspond to the GapC protein of S. pneumoniae .
  • the present invention provides GapC multiple epitope fusion proteins and polynucleotides encoding the same.
  • a multiple epitope fusion polypeptide comprising an amino acid sequence or a sequence with at least 80% sequence identity thereto, the amino acid sequence comprising the sequence shown as Figure 6 (SEQ ID NO: 22) but lacking an E. coli LipoF signal sequence depicted as residues 1 to 27 of Figure 6 (SEQ ID NO: 22).
  • the multiple epitope fusion polypeptide may comprise an amino acid sequence as set out above together with a signal sequence.
  • the signal sequence may comprise the amino acid sequence depicted at positions 1-27 of Figure 6 (SEQ ID NO: 22).
  • the multiple epitope fusion polypeptide comprises the amino acid sequence depicted in Figure 6 (SEQ ID NO: 22).
  • the invention is directed to polynucleotide sequences encoding the multiple epitope fusion polypeptide sequence described above or compliments thereof, as well as recombinant vectors comprising the polynucleotide, host cells comprising the recombinant vectors and methods of recombinantly producing the polypeptides.
  • the invention is directed to a vaccine composition
  • a vaccine composition comprising a pharmaceutically acceptable vehicle and a multiple epitope fusion polypeptide as described above.
  • the vaccine compositions comprise an adjuvant.
  • the invention is directed to a method of producing a vaccine composition comprising the steps of
  • the vaccine may be used in a method of treating or preventing a bacterial infection in a vertebrate subject, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition as described above.
  • the bacterial infection may be a streptococcal infection. Further, the bacterial infection may cause mastitis.
  • the polynucleotide may be used in a method of treating or preventing a bacterial infection in a vertebrate subject, the method comprising administering to the subject a therapeutically effective amount of a polynucleotide as described herein.
  • the bacterial infection may be a streptococcal infection. Further, the bacterial infection may cause mastitis.
  • a multiple epitope fusion polypeptide according to the 1 st aspect of the invention for the manufacture of a vaccine composition useful for treating or preventing a bacterial infection in a vertebrate subject.
  • the bacterial infection is a streptococcal infection.
  • the bacterial infection may cause mastitis.
  • the invention is directed to antibodies directed against the above multiple epitope fusion polypeptides.
  • the antibodies may be polyclonal or monoclonal.
  • the invention is directed to a method of detecting Streptococcus antibodies in a biological sample, comprising:
  • the invention is directed to an immunodiagnostic test kit for detecting Streptococcus infection.
  • the test kit comprises a multiple epitope fusion polypeptide as described herein and instructions for conducting the immunodiagnostic test.
  • GapC protein and “GapC plasmin binding protein” (used interchangeably herein) or a nucleotide sequence encoding the same, intends a protein or a nucleotide sequence, respectively, which is derived from a GapC gene found in a variety of Streptococcus species, including, without limitation certain strains of group A streptococci (Lottenbery, R.,. et al., (1987) Infect. Immun . 55 :1914-1918).
  • Figures 1 through 5 depict the isolated nucleotide sequences and isolated amino acid sequences of S. dysgalactiae (SEQ ID NO:11 and SEQ ID NO:12, respectively), S. agalactiae (SEQ ID NO:13 and SEQ ID NO:14, respectively), S. uberis (SEQ ID NO:15 and SEQ ID NO:16, respectively), S. parauberis (SEQ ID NO:17 and SEQ ID NO:18, respectively,), and S. iniae (SEQ ID NO:19 and SEQ ID NO:20, respectively).
  • a GapC protein as defined herein is not limited to the depicted sequences as subtypes of each of these Streptococcus species are known and variations in GapC proteins will occur between them.
  • gapC genes derived from S. dysgalactiae, S. agalactiae, S. uberis , and S. parauberis , are found in the plasmids pET15bgapC, pMF521c, pMF521 a, pMF521d, and pMF521e, respectively.
  • the derived protein or nucleotide sequences need not be physically derived from the gene described above, but may be generated in any manner, including for example, chemical synthesis, isolation (e.g., from S. dysgalactiae ) or by recombinant production, based on the information provided herein. Additionally, the term intends proteins having amino acid sequences substantially homologous (as defined below) to contiguous ammo acid sequences encoded by the genes, which display immunological and/or plasmin-binding activity.
  • nucleotide fragments of the gene that include at least about 8 contiguous base pairs, more preferably at least about 10-20 contiguous base pairs, and most preferably at least about 25 to 50, or more, contiguous base pairs of the gene, or any integers between these values. Such fragments are useful as probes and in diagnostic methods, discussed more fully below.
  • the terms also include those forms possessing, as well as lacking, a signal sequence, if such is present, as well as the nucleic acid sequences coding therefore. Additionally, the term intends forms of the GapC proteins which lack a membrane anchor region, and nucleic acid sequences encoding proteins with such deletions. Such deletions may be desirable in systems that do not provide for secretion of the protein. Furthermore, the plasmin-binding domains of the proteins, may or may not be present. Thus, for example, if the GapC plasmin-binding protein will be used to purify plasmin, the plasmin-binding domain will generally be retained. If the protein is to be used in vaccine compositions, immunogenic epitopes which may or may not include the plasmin-binding domain, will be present.
  • proteins in neutral form or in the form of basic or acid addition salts depending on the mode of preparation may involve free amino groups and basic salts may be formed with free carboxyls.
  • Pharmaceutically acceptable basic and acid addition salts are discussed further below.
  • the proteins may be modified by combination with other biological materials such as lipids (both those occurring naturally with the molecule or other lipids that do not destroy immunological activity) and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains, oxidation of sulfhydryl groups, glycosylation of amino acid residues, as well as other modifications of the encoded primary sequence.
  • substitutions intends deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.
  • particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 or 20-50 conservative or non-conservative amino acid substitutions, or any integer between these values, so long as the desired function of the molecule remains intact.
  • GapC proteins isolated from streptococci exhibit several variable regions in their amino acid sequences, located at amino acid positions 62 to 81; 102 to 112; 165 to 172; 248 to 271; and 286 to 305. These regions, which in S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis and S. iniae exhibit from 1 to 9 amino acid substitutions, are likely to be amenable to variation without substantially affecting immunogenic or enzymatic function.
  • streptococcal GapC protein intends a GapC plasmin-binding protein, as defined above, derived from a streptococcal species that produces the same, including, but not limited to S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis , and S. iniae .
  • a " S. dysgalactiae GapC protein” is a GapC plasmin-binding protein as defined above, derived from S. dysgalactiae .
  • an "S. agalactiae GapC protein” intends a gapC binding protein derived from S. agalactiae .
  • Wild type or “native” proteins or polypeptides refer to proteins or polypeptides isolated from the source in which the proteins naturally occur.
  • Recombinant polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
  • Synthetic polypeptides are those prepared by chemical synthesis.
  • isolated protein or polypeptide is a protein or polypeptide molecule separate and discrete from the whole organism with which the molecule is found in nature; or a protein or polypeptide devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
  • the term "functionally equivalent” intends that the amino acid sequence of a GapC plasmin-binding protein is one that will elicit a substantially equivalent or enhanced immunological response, as defined above, as compared to the response elicited by a GapC plasmin-binding protein having identity with the reference GapC plasmin-binding protein, or an immunogenic portion thereof.
  • epitope refers to the site on an antigen or hapten to which specific B cells and/or T cells respond.
  • the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
  • Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • Epitopes may include 3 to 5 amino acids, more preferably 5 to 10 amino acids, up to the full length of the reference molecule.
  • multiple epitope protein or polypeptide specifies a sequence of amino acids comprising an epitope as defined herein, which contains at least one epitope repeated two or more times within a linear molecule.
  • the repeating sequence need not be directly connected to itself, is not repeated in nature in the same manner and, further, may be present within a larger sequence which includes other amino acids that are not repeated.
  • the epitope sequence may either be an exact copy of a wild-type epitope sequence, or a sequence which is "functionally equivalent" as defined herein refers to a multiple epitope protein or polypeptide as defined herein that is produced by recombinant or synthetic methods.
  • a “fusion” or “chimeric” protein or polypeptide is one in which amino acid sequences from more than one source are joined. Such molecules may be produced synthetically or recombinantly, as described further herein (see the section entitled “Production of GapC Plasmin-Binding Proteins” infra ). Hence, the term “multiple epitope fusion protein or polypeptide” refers to a multiple epitope protein or polypeptide as defined herein which is made by either synthetic or recombinant means.
  • a multiple epitope fusion protein comprising the variable regions in the amino acid sequences of the GapC proteins referred to above may be produced.
  • the amino acid sequence for a representative GapC multiple epitope fusion protein, and a corresponding polynucleotide coding sequence, is depicted in Figures 6A-6C herein.
  • Methods for recombinantly producing the protein including a method for constructing the polyGap4 plasmid containing the chimeric coding sequence (diagramed in Figure 25 ) and a method for expressing the protein from the polyGap4 plasmid, are described in Examples 4 and 5 infra .
  • immunogenic protein or polypeptide refer to an amino acid sequence which elicits an immunological response as described herein.
  • An “immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of the GapC plasmin-binding protein in question, with or without the signal sequence, membrane anchor domain and/or plasmin-binding domain, analogs thereof, or immunogenic fragments thereof.
  • immunogenic fragment is meant a fragment of a GapC plasmin-binding protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art.
  • linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci.
  • conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra .
  • Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad.
  • Immunogenic fragments will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, and most preferably 25 or more amino acids, of the parent GapC plasmin-binding-binding protein molecule. There is no critical upper limit to the length of the fragment, which may comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes of GapC.
  • an "immunogenic composition” is a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest.
  • subunit vaccine composition is meant a composition containing at least one immunogenic polypeptide, but not all antigens, derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of intact pathogen cells or particles, or the lysate of such cells or particles.
  • a “subunit vaccine composition” is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof.
  • a subunit vaccine composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from the pathogen.
  • pharmaceutically acceptable or “pharmacologically acceptable” is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • an "immunological response" to a composition or vaccine is the development in the host of a cellular and/ or antibody-mediated immune response to the composition or vaccine of interest.
  • an "immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or ⁇ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host will display either a therapeutic or protective immunological response such that resistance of the mammary gland to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host and/or a quicker recovery time.
  • nucleic acid immunization is meant the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell, for the in vivo expression of an antigen, antigens, an epitope, or epitopes.
  • the nucleic acid molecule can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal and mucosal administration, or the like, or can be introduced ex vivo , into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule.
  • treatment refers to either (1) the prevention of infection or reinfection (prophylaxis), or (2) the reduction or elimination of symptoms of the disease of interest (therapy).
  • mastitis an inflammation of the mammary gland in mammals, including in cows, ewes, goats, sows, mares, and the like, caused by the presence of pathogenic microorganisms, such as S. uberis .
  • the infection manifests itself by the infiltration of phagocytic cells in the gland.
  • mastitis 4 clinical types are recognized: (1) peracute, associated with swelling, heat, plain, and abnormal secretion in the gland and accompanied by fever and other signs of systemic disturbance, such as marked depression, rapid weak pulse, sunken eyes, weakness and complete anorexia; (2) acute, with changes in the gland similar to those above but where fever, anorexia and depression are slight to moderate; (3) subacute, where no systemic changes are displayed and the changes in the gland and its secretion are less marked: and (4) subclinical, where the inflammatory reaction is detectable only by standard tests for mastitis.
  • Standard tests for the detection of mastitis include but are not limited to, the California Mastitis Test, the Wisconsin Mastitis Test, the Nagase test, the electronic cell count and somatic cell counts used to detect a persistently high white blood cell content in milk.
  • a somatic cell count of about 300,000 to about 500,000 cells per ml or higher, in milk will indicate the presence of infection.
  • a vaccine is considered effective in the treatment and/or prevention of mastitis when, for example, the somatic cell count in milk is retained below about 500,000 cells per ml.
  • vertebrate By the terms “vertebrate,” “subject,” and “vertebrate subject” are meant any member of the subphylum Chordata, including, without limitation, mammals such as cattle, sheep, pigs, goats, horses, and humans; domestic animals such as dogs and cats; and birds, including domestic, wild and game birds such as cocks and hens including chickens, turkeys and other gallinaceous birds; and fish.
  • the term does not denote a particular age. Thus, both adult and newborn animals, as well as fetuses, are intended to be covered.
  • a "nucleic acid" molecule can include, but is not limited to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • the term also captures sequences that include any of the known base analogs of DNA and RNA.
  • nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
  • isolated in the context of a polynucleotide intends that the polynucleotide is isolated from the chromosome with which it is normally associated, and is isolated from the complete genomic sequence in which it normally occurs.
  • Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • a "coding sequence” or a “nucleotide sequence encoding" a particular protein is a nucleotide sequence which is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • a "complementary" sequence is one in which the nitrogenous base at a given nucleotide position is the complement of the nitrogenous base appearing at the same position in the reference sequence.
  • the complement of adenosine is tyrosine, and vice versa; similarly, cytosine is complementary to guanine, and vice versa; hence, the complement of the reference sequence 5'-ATGCTGA-3' would be 5'-TACGACT-3'.
  • a “wild-type” or “native” sequence refers to polypeptide encoding sequences that are essentially as they are found in nature, e.g., the S. dysgalactiae GapC protein encoding sequences depicted in Figures 1A-1B (SEQ ID NO:12).
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • Recombinant host cells “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
  • Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
  • Homology refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.
  • nucleotide sequence identity is available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra .
  • degenerate variant is intended a polynucleotide containing changes in the nucleic acid sequence thereof, that encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the degenerate variant is derived.
  • similarity means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed “percent similarity” then can be determined between the compared polypeptide sequences.
  • Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • the heterologous region encodes a bacterial gene
  • the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria.
  • Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a vector is capable of transferring gene sequences to target cells (e.g., bacterial plasmid vectors, viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • vector construct all refer to an assembly which is capable of directing the expression of a sequence or gene of interest.
  • expression vector include cloning and expression vehicles, as well as viral vectors.
  • the expression cassettes described herein may be contained within a plasmid construct.
  • the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e . g ., a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • control elements refers collectively to transcription promoters, transcription enhancer elements, transcription termination sequences, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), translation termination sequences, upstream regulatory domains, ribosome binding sites and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. See e . g ., McCaughan et al. (1995) PNAS USA 92:5431-5435 ; Kochetov et al (1998) FEBS Letts. 440:351-355 .
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked” to the coding sequence.
  • control elements compatible with expression in a subject are those which are capable of effecting the expression of the coding sequence in that subject.
  • a control element such as a promoter "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a "host cell” is a cell which has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule.
  • a cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • a "biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
  • fluorescer refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.
  • labels which may be used include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH and ⁇ - ⁇ -galactosidase.
  • GapC protein is capable of eliciting an immune response in a vertebrate subject.
  • immunization of dairy cattle with the GapC protein of S. dysgalactiae conferred protection against experimental infection with this organism, and furthermore, conferred cross-protection against infection by S. uberis.
  • GapC is produced by a number of different streptococcus species. With the exception of several localized variable regions, the amino acid sequences of the GapC proteins produced by those strains are highly conserved. Therefore, it is desirable to construct multiple epitope GapC fusion proteins comprising antigenic determinants taken from both the highly conserved regions of GapC, and the unique regions of GapC proteins from several streptococcal species. Experiments performed in support have demonstrated that such a protein is capable of eliciting broad immunity against a variety of streptococcal infections while providing the additional economic advantage of minimizing the number of antigens present in the final formulation, and concomitantly reducing the cost of producing that formulation.
  • GapC multiple epitope fusion proteins which have general structural formula (A) x --(B) y --(C) z representing a linear amino acid sequence.
  • B is an amino acid sequence of at least five and not more than 1,000 amino acids of an antigenic determinant from a GapC protein
  • y is an integer of 2 or more.
  • a and C are each different from B, as well as being different from each other, and are independently an amino acid sequence of an antigenic determinant containing at least five and not more than 1,000 amino acids not immediately adjacent to B in nature.
  • x and z are each independently an integer of 0 or more, wherein at least one of x and z is 1 or more.
  • A, B, and C are antigenic determinants from the GapC proteins of one or more bacterial species.
  • A, B, and C are amino acid sequences comprising one or more antigenic determinants from the GapC protein of one or more of the following species of streptococcus: S. dysgalaetiae; S. agalactiae; S. uberis; S. parauberis , and S. iniae.
  • Figures 9 through 13 show plots of the following for the streptococcal GapC proteins employed: Kyte-Doolittle hydrophathy, averaged over a window of 7; surface probability according to Emini; chain flexibility according to Karplus-Schulz; antigenicity index according to Jameson-Wolf; secondary structure according to Gamier-Osguthorpe-Robson; secondary structure according to Chou-Fasman; and predicted glycosylation sites.
  • Figures 15 through 19 show plots of secondary structure according to Chou-Fasman for the aforementioned proteins.
  • One of skill in the art can readily use the information presented in Figures 9 through 13 and 15 to 19 in view of the teachings of the present specification to identity antigenic regions which may be employed in constructing a chimeric protein.
  • A, B, and/or C include one or more variable regions of the GapC proteins from more than one streptococcus species.
  • Figures 8A-8C show an amino acid sequence alignment which illustrates regions of homology and variability that exist among GapC proteins from S dysgalactiae, S. agalactiae, S. uberis, S. parauberis , and S. iniae. Amino acid sequences for the GapC proteins of S. pyogenes and S. equisimilis, S. pyogenes are also included.
  • several variable regions are located at amino acid positions 62 to 81; 102 to 112; 165 to 172; 248 to 271; and 286 to 305.
  • the multiple epitope fusion protein may also include spacer sequences interposed between A, B, and/or C.
  • the spacer sequences are typically amino acid sequences of from 1 to 1,000 amino acids, may be the same or different as A, B, or C, and may be the same or different as each other.
  • the protein may also include a signal sequence and/or a transmembrane sequence.
  • suitable signal sequences include the E. coli LipoF signal sequence, and the OmpF signal sequence.
  • suitable transmembrane sequences include those associated with LipoF and OmpF.
  • Gap4 The amino acid sequence of Gap4 (SEQ ID NO:22), a multiple epitope GapC fusion protein, is shown in Figures 6A-6C , as is the polynucleotide sequence which encodes it (SEQ ID NO:21).
  • Gap4 is a 47.905 kDa chimerie protein of 448 amino acids.
  • Residues 1 to 27 are identical to amino acid residues 1 to 27 of the E. coli LipoF signal sequence.
  • Residues 28 to 123 are identical to residues 1 to 96 of the S. dysgalactiae GapC protein.
  • Residues 124 (leucine) and 125 (glutamic acid) are spacer amino acids.
  • residues 126 to 165 are followed by residues 126 to 165, which are identical to residues 56 to 95 of S. parauberis as well as to the same residues of S. uberts .
  • Residue 166 isoleucine
  • Residues 167 to 208 are identical to residues 55 to 96 of the S. agalactiae GapC protein.
  • Residues 209 (threonine) and 210 (serine) are spacer amino acids.
  • Residues 211 to 448 are identical to residues 99 to 336 of the S. dysgalactiae GapC protein.
  • Gap4 has a cysteine residue present at the amino terminal end of the mature protein.
  • the LipoF signal sequence and cysteine residue are present to ensure that the chimeric molecule is efficiently secreted from the bacterial host cell and becomes bound to the host cell membrane via the lipid-moiety.
  • the protein may then be extracted from the cell surface via differential solubilization with a detergent such as Sarkosyl or TritonX-100® (see Example 5 infra ).
  • the GapC chimeric proteins or antigenic fragments thereof can be provided in subunit vaccine compositions.
  • the proteins or antibodies thereto can be used as diagnostic reagents to detect the presence of infection in a vertebrate subject.
  • the genes encoding the proteins can be cloned and used to design probes to detect and isolate homologous genes in other bacterial swains. For example, fragments comprising at least about 15-20 nucleotides, more preferably at least about 20-50 nucleotides, and most preferably about 60-100 nucleotides, or any integer between these values, will find use in these embodiments.
  • the vaccine compositions can be used to treat or prevent a wide variety of bacterial infections in vertebrate subjects.
  • vaccine compositions including GapC multiple epitope fusion proteins comprising antigenic determinants from S. dysgalactiae. S. uberis, S. parauberis, S. iniae , and/or group B streptococci (GBS) such as S. agalactiae
  • group B streptococci such as S. dysgalactiae
  • S. uberis and S. agalactiae are common bacterial pathogens associated with mastitis in bovine, equine, ovine and goat species.
  • group B streptococci such as S.
  • agalactiae are known to cause numerous other infections in vertebrates, including septicemia, meningitis, bacteremia, impetigo, arthritis, urinary tract infections, abscesses, spontaneous abortion etc.
  • vaccine compositions containing chimeric GapC proteins will find use in treating and/or preventing a wide variety of streptococcal infections.
  • GapC multiple epitope fusion proteins comprising antigenic determinants derived from other bacterial genera such as Staphylococcus, Mycobacterium, Escherichia, Pseudomonas, Nocardia, Pasteurella, Clostridium and Mycoplasma will find use for treating bacterial infections caused by species belonging to those genera.
  • chimeric GapC proteins can be used to treat and/or prevent a wide variety of bacterial infections in numerous species.
  • the GapC multiple epitope fusion proteins can be used in vaccine compositions either alone or in combination with other bacterial, fungal, viral or protozoal antigens. These other antigens can be provided separately or even as fusion proteins comprising the GapC chimeric protein fused to one or more of these antigens.
  • other immunogenic proteins from S. uberis such as the CAMP factor, hyaluronic acid capsule, hyaluronidase, R-like protein and plasminogen activator, can be administered with the chimeric GapC protein.
  • immunogenic proteins from other organisms involved in mastitis such as from the genera Staphylococcus, Corynebacterium, Pseudomonas, Nocardia, Clostridium, Mycobacteriurn, Mycoplasma, Pasteurella, Prototheca , other streptococci, coliform bacteria, as well as yeast, can be administered along with the GapC fusion proteins described herein to provide a broad spectrum of protection.
  • zooepidemicus Corynebacterium pyogenes, Pseudomonas aeruginosa, Nocardia asteroides, Clostridium perfringens, Escherichia coli, Enterobacter aerogenes and Klebsiella spp . can be provided along with the GapC plasmin-binding proteins.
  • chimeric proteins and active fragments and analogs derived from the same can be produced by recombinant methods as described herein. These recombinant products can take the form of partial protein sequences, full-length sequences, precursor forms that include signal sequences, or mature forms without signals.
  • GapC plasmin-binding protein DNA sequences used to construct the chimeric proteins can be isolated by a variety of methods known to those of skill in the art. See, e.g., Sambrook et al., supra . Methods for isolating, cloning and sequencing the gene sequences encoding GapC proteins from S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis , and S. iniae are detailed in Examples 1,2 and 3, infra .
  • polynucleotide sequences encoding the chimeric proteins are constructed using standard recombinant techniques including PCR amplification, restriction endonuclease digestion and ligation. See, e.g., Sambrook et al., supra . Methods for constructing Gap4 are detailed in Example 4, infra .
  • the DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned.
  • the DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756 ; Nambair et al. (1984) Science 223:1299 ; Jay et al. (1984) J. Biol. Chem. 259:6311 .
  • coding sequences for the desired proteins can be cloned into any suitable vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ⁇ ( E. coil ), pBR322 ( E. coli ), pACYC177 ( E. coil ), pKT230 (gram-negative bacteria), pGV1106 (grain-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-( E. coli gram-negative bacteria), pHV14 (( E.
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence. If a signal sequence is included, it can either be the native, homologous sequence, or a heterologous sequence.
  • the LipoF signal sequence is added to the amino-terminal region of the chimeric protein Gap4 to permit secretion of the protein after expression. See Examples 4E and 5, infra .
  • Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739 ; 4,425,437 ; 4,338,397 .
  • regulatory sequences which allow for regulation of expression of the protein sequences relative to the growth of the host cell may also be desirable. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al., supra ; DNA cloning, supra ; Nucleic Acid Hybridization, supra .
  • the expression vector is then used to transform an appropriate host cell.
  • mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ('MDBK”) cells, as well as others.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • baby hamster kidney (BHK) cells baby hamster kidney (BHK) cells
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • Madin-Darby bovine kidney ('MDBK) cells e.g., Madin-Darby bovine kidney ('MDBK) cells
  • bacterial hosts such as E. coli, Bacillus subtilis and
  • Useful yeast hosts include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polyrnorpha, Kluyveromyces friagilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica .
  • Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda , and Trichoplusia ni .
  • the proteins may be produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into the growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the proteins may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984 ) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254 , for solid phase peptide synthesis techniques; and M.
  • the chimeric GapC plasmin-binding proteins, or their fragments can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370 . If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffmity chromatography, using known procedures.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • Serum from the immunized animal is collected and treated according to known procedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370
  • Monoclonal antibodies to the chimeric GapC plasmin-binding proteins and to the fragments thereof can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by using hybridoma technology is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980 ); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981 ); Kennett et al., Monoclonal Antibodies (1980 ); see also U.S.
  • Panels of monoclonal antibodies produced against the chimeric GapC plasmin-binding proteins, or fragments thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes.
  • the GapC multiple epitope fusion proteins can be formulated into vaccine compositions, either alone or in combination with other antigens, for use in immunizing subjects as described below. Methods of preparing such formulations are described in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18 Edition, 1990 .
  • the vaccines are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • the active immunogenic ingredient is generally mixed with a compatible pharmaceutical vehicle, such as, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof,
  • a compatible pharmaceutical vehicle such as, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.
  • Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation.
  • Adjuvants may include for example, muramyl dipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art.
  • DDA dimethyldioctadecyl ammonium bromide
  • the chimeric GapC plasmin-binding protein may be linked to a carrier in order to increase the immunogenicity thereof.
  • Suitable carriers include large, slowly metabolized macromolecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.
  • proteins including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art
  • polysaccharides such as sepharose, agarose, cellulose, cellulose beads and the like
  • polymeric amino acids such as polyglutamic acid, polylysine, and the like
  • the chimeric GapC plasmin-binding proteins may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone.
  • a sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate.
  • Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.
  • Suitable carriers for the chimeric GapC plasmin-binding proteins include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S. Patent No. 5,071,651 . Also useful is a fusion product of a viral protein and the subject chimeric proteins made by methods disclosed in U.S. Patent No. 4,722,840 . Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the proteins may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.
  • the chimeric GapC plasmin-binding proteins may be formulated into vaccine compositions in either neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an immune response in a subject to which the composition is administered.
  • a "therapeutically effective amount” would preferably be an amount that enhance resistance of the mammary gland to new infection and/or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered somatic cell count in milk from the infected quarter.
  • the ability of the composition to retain or bring the somatic cell count (SCC) in milk below about 500,000 cells per ml, the threshold value set by the International Dairy Federation, above which, animals are considered to have clinical mastitis, will be indicative of a therapeutic effect.
  • the exact amount is readily determined by one skilled in the art using standard tests.
  • the chimeric GapC plasmin-binding protein concentration will typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate.
  • 5 to 500 ⁇ g of active ingredient per ml of injected solution should be adequate to raise an immunological response when a dose of 1 to 3 ml per animal is administered.
  • the vaccine is generally administered parenterally, usually by intramuscular injection.
  • Other modes of administration such as subcutaneous, intraperitoneal and intravenous injection, are also acceptable.
  • the quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • the subject is immunized by administration of the vaccine in at least one dose, and preferably two doses.
  • the animal may be administered as many doses as is required to maintain a state of immunity to infection.
  • Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations.
  • the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides.
  • Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.
  • Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like.
  • These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.
  • Diluents such as water, aqueous saline or other known substances can be employed
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • Controlled or sustained release formulations are made by incorporating the protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures.
  • carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures.
  • the chimeric GapC plasmin-binding proteins can also be delivered using implanted mini-pumps, well known in the art.
  • the chimeric GapC plasmin-binding proteins can also be administered via a carrier virus which expresses the same.
  • Carrier viruses which will find use include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus.
  • vaccinia virus recombinants expressing the novel proteins can be constructed as follows. The DNA encoding the particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome.
  • the resulting TK recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
  • nucleotide sequences (and accompanying regulatory elements) encoding the subject chimeric GapC plasmin-binding proteins can be administered directly to a subject for in vivo translation thereof.
  • gene transfer can be accomplished by transfecting the subjects cells or tissues ex vivo and reintroducing the transformed material into the host.
  • DNA can be directly introduced into the host organism, i.e., by injection (see International Publication No. WO/90/11092 ; and Wolff et al. (1990) Science 247:1465-1468 ).
  • Liposome-mediated gene transfer can also be accomplished using known methods. See, e.g., Hazinski. et al. (1991) Am.
  • Targeting agents such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to infection.
  • the chimeric GapC plasmin-binding proteins may also be used as diagnostics to detect the presence of reactive antibodies of streptococcus, for example S. dysgalactiae , in a biological sample in order to determine the presence of streptococcus infection.
  • the presence of antibodies reactive with chimeric GapC plasmin-binding proteins can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound antibody in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • a solid support is first reacted with a solid phase component (e.g., one or more chimeric GapC plasmin-binding proteins) under suitable binding conditions such that the component is sufficiently immobilized to the support.
  • a solid phase component e.g., one or more chimeric GapC plasmin-binding proteins
  • immobilization of the antigen to the support can be enhanced by first coupling the antigen to a protein with better binding properties.
  • Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
  • molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like.
  • Such molecules and methods of coupling these molecules to the antigens are well known to those of ordinary skill in the art. See, e.g., Brinkley, M.A. Bioconjugate Chem. (1992) 3:2-13 ; Hashida et al., J. Appl. Biochem. (1984) 6:56-63 and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30:117-124 .
  • any non-immobilized solid-phase components are removed from the support by washing, and the support-bound component is then contacted with a biological sample suspected of containing ligand moieties (e.g., antibodies toward the immobilized antigens) under suitable binding conditions.
  • a biological sample suspected of containing ligand moieties e.g., antibodies toward the immobilized antigens
  • a secondary binder moiety is added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound ligand. The presence of the secondary binder can then be detected using techniques well known in the art.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a chimeric GapC plasmin-binding protein.
  • a biological sample containing or suspected of containing anti-chimeric GapC plasmin-binding protein immunoglobulin molecules is then added to the coated wells.
  • the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added.
  • the secondary binding molecule is allowed to react with any captured sample antibodies, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • the presence of bound anti-chimeric GapC plasmin-binding antigen ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands.
  • a secondary binder comprising an antibody directed against the antibody ligands.
  • a number of anti-bovine immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art.
  • An appropriate enzyme substrate is then used to generate a detectable signal.
  • competitive-type ELISA techniques can be practiced using methods known to those skilled in the art.
  • Assays can also be conducted in solution, such that the chimeric GapC plasmin-binding proteins and antibodies specific for those proteins form complexes under precipitating conditions.
  • chimeric GapC plasmin-binding proteins can be attached to a solid phase particle (e.g., an agarose bead or the like) using coupling techniques known in the art, such as by direct chemical or indirect coupling.
  • the antigen-coated particle is then contacted under suitable binding conditions with a biological sample suspected of containing antibodies for the chimeric GapC plasmin-binding proteins.
  • Cross-linking between bound antibodies causes the formation of particle-antigen-antibody complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation.
  • the reaction mixture can be analyzed to determine the presence or absence of antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.
  • an immunoaffinity matrix can be provided, wherein a polyclonal population of antibodies from a biological sample suspected of containing anti-chimeric GapC plasmin-binding molecules is immobilized to a substrate.
  • an initial affinity purification of the sample can be carried out using immobilized antigens.
  • the resultant sample preparation will thus only contain anti-streptococcus moieties, avoiding potential nonspecific binding properties in the affinity support.
  • a number of methods of immobilizing immunoglobulins (either intact or in specific fragments) at high yield and good retention of antigen binding activity are known in the art. Not being limited by any particular method, immobilized protein A or protein G can be used to immobilize immunoglobulins.
  • labeled chimeric GapC plasmin-binding proteins are contacted with the bound antibodies under suitable binding conditions.
  • the presence of bound antigen can be determined by assaying for label using methods known in the art.
  • antibodies raised to the chimeric GapC plasmin-binding proteins can be used in the above-described assays in order to detect the presence of antibodies to the proteins in a given sample. These assays are performed essentially as described above and are well known to those of skill in the art.
  • the above-described assay reagents including the chimeric GapC plasmin-binding proteins, or antibodies thereto, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above.
  • the kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.
  • a deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, under the provisions of the Budapest Treaty. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. The designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.
  • a clinical S. dysgalactiae isolate from a case of bovine mastitis was obtained from the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209), and was used as a source of DNA. The organism was routinely grown on TSA sheep blood agar plates (PML Microbiologicals, Mississauga, Ontario) at 37° C for 18 hours, or in Todd-Hewitt broth (Oxoid Ltd., Hampshire, England) supplemented with 0.3% yeast extract (THB-YE) at 37° C, 5% CO 2 .
  • Chromosomal DNA was prepared from S. dysgalactiae grown in 100 ml of THB-YE supplemented with 20 mM glycine for approximately 6 hours, until an A 600 of 0.8 to 1.0 was reached.
  • Cells were harvested and re-suspended in 50 mM EDTA, 50 mM Tris-HCl, 0.5% Tween-20® (Sigma, St. Louis, MO) and supplemented with RNase A (200 mg/ml), proteinase K (20 mg/ml), lysozyme (100 mg/ml) and mutanolysin (100 mg/ml). (all enzymes purchased from SIGMA, St. Louis, MO).
  • guanidine hydrochloride and Twee-2®, pH 5.5 were mixed with the lysate to give a final concentration of 0.8 M and 5%, respectively. This mixture was incubated at 50° C for 30 minutes.
  • the chromosomal DNA was then purified using a Qiagen genomic-tip 100g (Qiagen, Santa Clarita, California) and precipitated using 0.7 volumes of isopropanol. The resulting pellet was washed in 70% ethanol and re-suspended in 0.5 ml 10 mM Tris-HCl, pH 8.8.
  • Chromosomal DNA from S. agalactiae, S. uberis and, S. parauberis was isolated essentially as described above, from strains designated ATCC 27541, 9927, and 13386, respectively. Chromosomal DNA from S. iniae was also isolated as above from a strain designated 9117 obtained from Mount Sinai Hospital, University of Toronto, Canada.
  • the polynucleotide sequences encoding GapC from S. dysgalactiae, S. uberis, S. parauberis, S. agalactiae and S. iniae were initially isolated from chromosomal DNA by PCR amplification.
  • the primers used to PCR-amplify the gapC genes from all species were gapC1 (SEQ ID NO:1) and gapC1r (SEQ ID NO:2), shown in Table 1.
  • underlining denotes nucleotides added to the original sequences (i.e., nucleotides added to the 5' end of the original sense strand sequence and to the 3' end of the original anti-sense strand sequence, respectively, of the gapC coding region being amplified ), and bolding indicates the location of restriction endonuclease recognition sites.
  • PCR was carried out using Vent DNA polymerase (New England Biolabs, Mississauga, ON, Canada).
  • a reaction mixture containing 0.2 ⁇ g of genomic DNA, 1pM of each of the preceding primers, 100 pM each of dATP, dTTP, dCTP and dGTP, 10mM Tris HCL, pH9; 1.5mM MgCl 2 , 50mM HCL, 1.5 units Taq DNA polymerase (Pharmacia, Quebec, Canada) was incubated for 40 amplification cycles of 40 seconds at 94°C, 40 seconds at 55 °C, and 1 minute, 20 seconds at 72°C, and then for a single cycle of 10 minutes at 72°C.
  • the resulting PCR reaction products were then digested with NdeI and BamHI .
  • the fragment was cloned directly into the same sites of pET15b (Novagen, Madison, Wisconsin) after the plasmid was digested with the same enzymes.
  • the resulting construct was denominated pET15bgapC.
  • S. agalactiae S. uberis, S. parauberis and S.
  • the plasmids containing the S. agalactiae, S. uberis, S. parauberis and S. iniae sequences were designated pMF521c-inv, pMF521a-inv, pMF521d-inv, and pMF521e-inv, respectively.
  • Table 1 Sequence Identification Numbers and Corresponding Nucleotide and Amino Acid Sequences SEQ ID NO.
  • genes isolated and cloned in the preceding examples were sequenced using fluorescence tag terminators on an ABI 373 DNA automatic sequencer (Applied Biosystems, Emeryville, California) at the Plant Biotechnology Institute (PBI, Saskatoon, Saskatchewan, Canada).
  • a chimeric gapC gene composed of sequences from S. dysgalactiae, S. parauberis, and S. agalactiae was constructed in a three-step process using pAA556, a standard tac-inducible expression plasmid derived from the plasmid pGH432 that contains the signal sequence from the E. Coli ompF gene.
  • the partial gapC gene sequences used to construct the chimeric gene were prepared by PCR amplification of selected polynucleotide sequences from the genomic gapC genes isolated above, using the primers Gap-1 through Gap-8.
  • the primer sequences are depicted in Table 1.
  • the chimeric gene sans the omp F signal sequence, was then excised from pAA556 and inserted into the plasmid pAA555, a pGH432 derivative that is a standard tac -inducible expression plasmid containing the signal sequence from the E. coli omp F gene.
  • the first 288 bases of the S . dysgalactiae gapC gene were PCR amplified using the primers Gap-1 and Gap-2.
  • PCR amplification was carried out as follows: 1.6 ⁇ g of template DNA was combined in a reaction mixture containing 20 pM each of primer Gap-1 (SEQ ID NO:1) and Gap-2 (SEQ ID NO:2), 200 ⁇ m each of dATP, dCTP, dGTP and dTTP, 2.5mM MgSO 4 , PCR Buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), and 1 unit Taq DNA polymerase (Pharmacia, Quebec, Canada). The mix was amplified for 1 cycle of 1 minute at 95°C, then for 29 cycles of 1 minute at 95°C, 1 minute at 55°C, and 30 seconds at 72°C, and finally for 1 cycle of 10 minutes at 4°C.
  • the amplification product was then digested with BamHI and NcoI and inserted into the same sites of an pAA556 vector.
  • the resulting plasmid construct designated pPolyGap.1, is illustrated in Figure 21 .
  • a PCR product representing bases 170-285 of the S. parauberis gapC gene was then obtained using the primers Gap-3 (SEQ ID NO:5) and Gap-4 (SEQ ID NO:6). This product codes for an amino acid sequence identical to the corresponding amino acid sequence found in the S. uberis gapC gene. PCR amplification was carried out essentially as above, except using 2 ⁇ g of template DNA.
  • the S. parauberis PCR product and the pPolyGap1plasmid were both digested with Xho1 and Nco1, and the PCR product was ligated into the corresponding sites in the vector.
  • This construct, called pPolyGap.2, is illustrated in Figure 22 .
  • Nucleotides 166-288 of the S. agalactiae gapC gene were amplified using PCR primers Gap-5 (SEQ ID NO:7) and Gap-6 (SEQ ID NO:8) as in Example 4B above.
  • pPolyGap3 is diagramed in Figure 23 .
  • the final step in constructing the chimeric gene involved the insertion of the remaining S. dysgalactiae gapC sequence (nucleotides 295-1011) in-frame and immediately downstream of the S. agalactiae sequence.
  • the S. dysgalactiae sequence was first PCR amplified using the primers Gap-7 (SEQ ID NO:9) and Gap-8 (SEQ ID NO:10) as in Example 4A above.
  • the amplification product was then digested with the enzyme GamHi/NcoI, as was the pPolyGap.3 vector, and the fragment was then ligated into the corresponding vector sites.
  • the chimeric gapc gene constructed in the preceding steps was excised from pAA556 by digestion with BamH1 and NcoI and inserted into the plasmid pAA555 digested with the same enzymes.
  • pAA555 is identical to pAA556 except that the former plasmid contains the LipoF signal sequence, and provides for the addition of a cysteine at the amino terminal end of the mature GapC protein.
  • the N-terminal cysteine was added to insure the chimeric protein's efficient secretion of from the cell and binding to the membrane via the lipid-moiety.
  • the coding sequence of the PolyGap4 plasmid construct is shown in Figure 25 .
  • PolyGap4 is used to transform E. coli J5 in the presence of polyethlene glycol (Kurien and Scofield (1995) BioTeclaniques 18 :1023-1026).
  • the transformed cells carrying pPolyGap4 are grown to logarithmic phase in LB media at 37°C with shaking. Expression of the chimeric GapC protein is then induced by adding IPTG to a final concentration of 1mM and incubating the cells at 37°C for an additional 4 hours.
  • the chimeric GapC protein is then extracted from the cell surface by differential solubilization.
  • the cells are collected by centrifugation and re-suspended in a volume of resuspension buffer (0.85% NaCl solution containing 0.6% sarkosyl) equal to 1/10th the original culture volume.
  • the suspension is incubated at room temperature for 30 minutes with gentle shaking.
  • the cells are collected by cenfrifuation and the supernatant containing the chimeric GapC protein is passed through a 0.2 ⁇ m membrane filter. Aliquots of the sterile supernatant are analyzed by SDS-PAGE and Western blots using a rabbit anti-GapC polyclonal antibody.
  • Vaccines were formulated in such a fashion that they contained 100 ⁇ g/ml of purified chimeric GapC protein in the oil-based adjuvant VSA3 (VIDO, Saskatoon, Saskatchewan, Canada).
  • VSA3 is a combination of Emulsigen PlusTM (MVP Laboratories, Ralston, Iowa) and dimethyldioctadecyl ammonium bromide (Kodak, Rochester, New York).
  • Non-lactating Holstein cows with no history of S. dysgalactiae infection are obtained. Two weeks prior to vaccination, all animals are treated with 300 mg of Cephapirin per quarter (Cepha-dryTM, Ayerst Laboratories, Montreal, Canada), in order to resolve any pre-existing udder infection prior to the vaccination step.
  • Cephapirin per quarter Cepha-dryTM, Ayerst Laboratories, Montreal, Canada
  • Groups of experimental animals are immunized subcutaneously with two doses of vaccines containing the chimeric GapC protein or a placebo with a three-week interval between immunizations.
  • Ten days to two weeks following the second immunization animals are exposed to 500-1,000 colony forming units of S. dysgalactiae delivered into three quarters with an udder infusion cannula. The fourth quarter on each animal serves as an un-infective control.
  • GapC-specific antibodies in bovine serum are measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre plates (NUNC, Naperville, Illinois) are coated by adding 0.1 microgram per well purified chimeric protein in 50mM sodium carbonate buffer, pH 9.6, incubated overnight at 4° C. The liquid is removed and the wells are blocked with 3% bovine serum albumin for 1 hr at 37° C. Serial dilutions of bovine serum (from 1:4 to 1:64,000) are added to the wells and incubated for 2 hours at room temperature.
  • ELISA enzyme-linked immunosorbent assay
  • the wells are aspirated, washed and incubated with 100 ⁇ l of alkaline phosphatase-conjugated goat anti-bovine IgG (Kirkgaard & Perry Laboratories Inc., Gaithersburg, Maryland) for 1 hr at room temperature.
  • the wells are washed again, and 100 ⁇ l of p-nitrophenol phosphate (Sigma, St. Louis, Missouri) is added as a substrate to detect alkaline phosphatase activity.
  • the absorbance at 405 nanometers is recorded following 1 hr incubation with the substrate at room temperature.
  • Bacteria are enumerated by spreading serial dilutions (10 0 to 10 -6 ) directly onto TSA sheep blood agar plates followed by overnight incubation at 37°C, 5% CO 2 . Colonization is defined as >500 cfu/ml of the challenge organism recovered.
  • Inflammatory response is measured as a function of mammary gland somatic cell count i.e., lymphocytes, neutrophils, and monocytes). Somatic cell counts are measured using standard techniques recommended by Agriculture and AgriFood Canada (IDF50B (1985): Milk and Milk Products--Methods of Sampling in a Coulter counter). Samples are read within 48 hours of collection and fixation, at days 1 through 7 post challenge.
  • the numbers of somatic cells present in the gland are determined on each day post challenge.

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Description

    Technical Field
  • The present invention relates generally to bacterial antigens and genes encoding the same. More particularly, the present invention pertains to the construction of a chimeric plasmin binding protein gene.
    • Background
  • Mastitis, an infection of the mammary gland usually caused by bacteria or fungus, results in major economic losses to the dairy industry yearly. Among the bacterial species most commonly associated with mastitis are various species of the genus Streptococcus, including S. aureus, S. uberis, (untypeable), S. agalactiae (Lancefield group B), S. dysgalactiae (Lancefleld group C), S. zooepidemicus, and the Lancefield groups D, G., L and N streptococci. Some of those species are contagious (e.g. S. agalactiae), while others are considered environmental pathogens (e.g. S. dysgalactiae and S. uberis. The environmental pathogen S. uberis is responsible for about 20% of all clinical cases of mastitis (Bramley, A.J. and Dodd, F.H. (1984) J. Dairy Res. 51:481-512; Bramley, A.J. (1987) Animal Health Nutrition 42:12-16; Watts, J.L. (1988) J. Dairy Sci.71:1616-1624); it is the predominant organism isolated from mammary glands during the non-lactating period (Bramley, A.J. (1984) Br. Vet. J. 140:328-335; Brainley and Dodd (1984) J. Dairy Res. 51:481-512; Oliver, S.P. (1988) Am. J. Vet. Res. 49:1789-1793).
  • Mastitis resulting from infection with S. uberis is commonly subclinical, characterized by apparently normal milk with an increase in somatic cell counts due to the influx of leukocytes. The chemical composition of milk is changed due to suppression of secretion with the transfer of sodium chloride and bicarbonate from blood to milk, causing a shift of pH to a more alkaline level. S. uberis mastitis may also take the form of an acute clinical condition, with obvious signs of disease such as clots or discoloration of the milk and swelling or hardness of the mammary gland. Some cases of the clinical disease can be severe and pyrexia may be present. For a review of the clinical manifestations of S. ubenis mastitis, see, Bramley (1991) Mastitis: physiology or pathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and A. W. Hill (ed.), New insights into the pathogenesis of mastitis. Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The mastitis complex-A brief summary. p. 1-3. In Bovine Mastitis. Lea & Febiger, Philadelphia
  • Conventional antibacterial control methods such as teat dipping and antibiotic therapy are effective in the control of many types of contagious mastitis, but the environmental organisms typically found in all dairy barns are often resistant to such measures. Vaccination is therefore an attractive strategy to prevent infections of the mammary glands, and has been shown to be beneficial in the case of some contagious mastitis pathogens.
  • The literature is limited regarding vaccination studies with S. dysgalactiae and S. uberis, and variable results have been observed. In some cases, immunization has resulted in increased sensitivity to the specific organism and in other cases strain-specific protection has been obtained.
  • For example, previous studies have shown that primary infection with S. uberis can considerably reduce the rate of infection following a second challenge with the same strain (Hill, A.W. (1988) Res. Vet. Sci. 44:386-387). Local vaccination with killed S. uberis protects the bovine mammary gland against intramammary challenge with the homologous strain (Finch et al. (1994) Infect. Immun. 62:3599-3603). Similarly, subcutaneous vaccination with live S. uberis has been shown to cause a dramatic modification of the pathogenesis of mastitis with the same strain (Hill et al. (1994) FEMS Immunol. Med. Microbiol. 8:109-118). Animals vaccinated in this way shed fewer bacteria in their milk and many quarters remain free of infection.
  • Nonetheless, vaccination with live or attenuated bacteria can pose risks to the recipient. Further, it is clear that conventional killed vaccines are in general largely ineffective against S. uberis and S. agalactiae, either due to lack of protective antigens on in vitro-grown cells or masking of these antigens by molecular mimicry.
  • The current lack of existing mastitis vaccines against S. agalactiae or the contagious streptococcus strains is due at least in part to a lack of knowledge regarding the virulence determinants and protective antigens produced by those organisms which are involved in invasion and protection of the mammary gland (Collins et al. (1988) J. Dairy Res. 55:25-32; Leigh et al. (1990) Res. Vet. Sci. 49: 85-87; Marshall et al. (1986) J. Dairy Res. 53: 507-514).
  • S. dysgalactiae is known to bind several extracellular and plasma-derived proteins such as fibronectin, fibrinogen, collagen, alpha-II-macroglobulin, IgG, albumin and other compounds. The organism also produces hyaluronidase and fibrinolysin and is capable of adhering to and invading bovine mammary epithelial cells. However, the exact roles of the bacterial components responsible for these phenotypes in pathogenesis is not known.
  • Similarly, the pathogenesis of S. uberis infection is poorly understood. Furthermore, the influence of S. uberis virulence factors on host defense mechanisms and mammary gland physiology is not well defined. Known virulence factors associated with S. uberis include a hyaluronic acid capsule (Hill, A.W. (1988) Res. Vet. Sci. 45:400-404), hyaluronidase (Schaufuss et al. (1989) Zentralbl. Bakteriol. Ser. A 271:46-53), R-like protein (Groschup, M.H. and Timoney, J.F. (1993) Res. Vet. Sci. 54:124-126), and a cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A 249:190-194), R-like protein, plasminogen activator and CAMP factor. However, very little is known of their roles in pathogenicity.
  • The use of virulence determinants from Streptococcus as immunogenic agents has been proposed. For example, the CAMP factor of S. uberis has been shown to protect vertebrate subjects from infection by that organism ( Jiang, U.S. Patent No. 5,863,543 ).
  • The γ antigen of the group B Streptococci strain A909 (ATCC No. 27591) is a component of the c protein marker complex, which additionally comprises an α and β subunit ( Boyle, U.S. Patent No. 5,721,339 ). Subsets of serotype Ia, II, and virtually all serotype Ib cells of group B streptococci, have been reported to express components of the c protein. Use of the γ subunit as an immunogenic agent against infections by Lancefield Group B Streptococcus infection has been proposed. However, its use to prevent or treat bacterial infections in animals, including mastitis in cattle, has not been studied.
  • A GapC plasmin binding protein from a strain of Group A Streptococcus has previously been identified and characterized, and its use in thrombolytic therapies has been described ( Boyle, et al., U.S. Patent No. 5,237,050 ; Boyle, et al., U.S. Patent No. 5,328,996 ). However, the use of GapC as an immungenic agent to treat or prevent mastitis was neither described nor suggested.
  • The group A streptococcal M protein is considered to be one of the major virulence factors of this organism by virtue of its ability to impede attack by human phagocytes (Lancefield, R.C. (1962) J. Immunol. 89:307-313). The bacteria persist in the infected tissue until antibodies are produced against the M molecule. Type-specific antibodies to the M protein are able to reverse the antiphagocytic effect of the molecule and allow efficient clearance of the invading organism.
  • M proteins are one of the key virulence factors of Streptococcus pyogenes, due to their involvement in mediating resistance to phagocytosis (Kehoe, M.A. (1991) Vaccine 9:797-806) and their ability to induce potentially harmful host immune responses via their superantigenicity and their capacity to induce host-cross-reactive antibody responses (Bisno, A.L. (1991) New Engl. J. Med. 325:783-793; Froude et al. (1989) Curr. Top. Microbiol. Immunol. 145:5-26; Stollerman, G.H. (1991) Clin. Immunol. Immunopathol. 61:131-142).
  • However, obstacles exist to using intact M proteins as vaccines. The protein's opsonic epitopes are extremely type-specific, resulting in narrow, type-specific protection. Further, some M proteins appear to contain epitopes that cross react with tissues of the immunized subject, causing a harmful autoimmune response (See e.g., Dale, J.L. and Beached, G.H. (1982) J. Exp. Med 156:1165-1176; Dale, J.L. and Beached, G.H. (1985) J. Exp. Med. 161:113-122; Baird, R.W., Bronze, M.S., Drabs, W., Hill, H.R., Veasey, L.G. and Dale, J.L. (1991) J. Immun. 146:3132-3137; Bronze, M.S. and Dale, J.L. (1993) J. Immun 151:2820-2828; Cunningham, M.W. and Russell, S.M. (1983) Infect. Immun. 42:531-538).
  • An octavalent M protein vaccine has been constructed and was tested for protective immunogenicity against multiple serotypes of group A streptococci infection in rabbits. However, the immune response obtained was serotype-specific, conferring protection only against those bacterial strains exhibiting the M protein epitopes present in the chimeric protein (Dale, J.B., Simmons, M. , Chiang, E.C., and Chiang, E.Y. (1996) Vaccine 14:944-948).
  • Chimeric proteins containing three different fibronectin binding domains (FNBDs) derived from fibronectin binding proteins of S. dysgalactiae and Staphylococcus aureus have been expressed on the surface of Staph. carnosus cells. In the case of one of these proteins, intranasal immunizations with live recombinant Staph. carnosus cells expressing the chlineric protein on their surface resulted in an improved antibody response to a model immunogen present within the chimeric surface protein.
  • A chimeric Protein G molecule (a type III Fc binding protein specific for the Fc region of all subclasses of IgG antibody molecules) is known, but its use as an immunogenic agent has not been described or suggested ( Bjorck, et al. (1992) U.S. Patent No. 5,108,894 ).
  • Until now, the protective capability of GapC multiple epitope fusion proteins has not been studied.
  • WO 98/18930 describes antigenic peptides from Steptococcus pneumoniae, including a peptide which may correspond to the GapC protein of S. pneumoniae.
    • Summary of the Invention
  • Accordingly, the present invention provides GapC multiple epitope fusion proteins and polynucleotides encoding the same. According to the present invention, we provide a multiple epitope fusion polypeptide comprising an amino acid sequence or a sequence with at least 80% sequence identity thereto, the amino acid sequence comprising the sequence shown as Figure 6 (SEQ ID NO: 22) but lacking an E. coli LipoF signal sequence depicted as residues 1 to 27 of Figure 6 (SEQ ID NO: 22).
  • The multiple epitope fusion polypeptide may comprise an amino acid sequence as set out above together with a signal sequence. The signal sequence may comprise the amino acid sequence depicted at positions 1-27 of Figure 6 (SEQ ID NO: 22).
  • In some embodiments, the multiple epitope fusion polypeptide comprises the amino acid sequence depicted in Figure 6 (SEQ ID NO: 22).
  • We describe a multiple epitope fusion polypeptide which comprises the amino acid sequence depicted in Figure 6 (SEQ ID NO:22).
  • In yet further embodiments, the invention is directed to polynucleotide sequences encoding the multiple epitope fusion polypeptide sequence described above or compliments thereof, as well as recombinant vectors comprising the polynucleotide, host cells comprising the recombinant vectors and methods of recombinantly producing the polypeptides.
  • In another embodiment, the invention is directed to a vaccine composition comprising a pharmaceutically acceptable vehicle and a multiple epitope fusion polypeptide as described above. In certain embodiments, the vaccine compositions comprise an adjuvant.
  • In still a further embodiment, the invention is directed to a method of producing a vaccine composition comprising the steps of
    1. (1) providing the multiple epitope fusion polypeptide; and
    2. (2) combining the polypeptide with a pharmaceutically acceptable vehicle.
  • The vaccine may be used in a method of treating or preventing a bacterial infection in a vertebrate subject, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition as described above.
  • The bacterial infection may be a streptococcal infection. Further, the bacterial infection may cause mastitis.
  • The polynucleotide may be used in a method of treating or preventing a bacterial infection in a vertebrate subject, the method comprising administering to the subject a therapeutically effective amount of a polynucleotide as described herein.
  • The bacterial infection may be a streptococcal infection. Further, the bacterial infection may cause mastitis.
  • According to another aspect of the invention, we provide for the use of a multiple epitope fusion polypeptide according to the 1st aspect of the invention for the manufacture of a vaccine composition useful for treating or preventing a bacterial infection in a vertebrate subject.
  • In some embodiments, the bacterial infection is a streptococcal infection. The bacterial infection may cause mastitis.
  • In further embodiments, the invention is directed to antibodies directed against the above multiple epitope fusion polypeptides. The antibodies may be polyclonal or monoclonal.
  • In another embodiment, the invention is directed to a method of detecting Streptococcus antibodies in a biological sample, comprising:
    1. (a) reacting said biological sample with a multiple epitope fusion polypeptide under conditions which allow said Streptococcus antibodies, when present in the biological sample, to bind to said sequence to form an antibody/antigen complex; and
    2. (b) detecting the presence or absence of said complex, and thereby detecting the presence or absence of Streptococcus antibodies in said sample.
  • In still a further embodiment, the invention is directed to an immunodiagnostic test kit for detecting Streptococcus infection. The test kit comprises a multiple epitope fusion polypeptide as described herein and instructions for conducting the immunodiagnostic test.
  • These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.
    • Brief Description of the Figures
    • Figures 1A-1B depict the isolated nucleotide sequence and deduced amino acid sequence of the gapC gene for S. dysgalactiae (SEQ ID NO:11 and SEQ ID NO:12). In the figure, the asterisk represents a stop codon, and the underlined regions represent nucleotide sequences complementary to the primers used to isolate the genes from the bacterial chromosomes.
    • Figures 2A-2B depict the isolated nucleotide sequence and deduced amino acid sequence of the gapC gene for S. agalactiae (SEQ ID NO:13 and SEQ ID NO:14). In the figure, the asterisk represents a stop codon, and the underlined regions represent nucleotide sequences complementary to the primers used to isolate the genes from the bacterial chromosomes.
    • Figures 3A-3B depict the isolated nucleotide sequence and deduced amino acid sequence of the gapC gene for S. uberis (SEQ ID NO:15) and SEQ ID NO:16). In the figure, the asterisk represents a stop codon, and the underlined regions represent nucleotide sequences complementary to the primers used to isolate the genes from the bacterial chromosomes.
    • Figures 4A-4B depict the isolated nucleotide sequence and deduced amino acid sequence of the gapC gene for S. parauberis (SEQ ID NO:17 and SEQ ID NO:18). In the figure, the asterisk represents a stop codon, and the underlined regions represent nucleotide sequences complementary to the primers used to isolate the genes from the bacterial chromosomes.
    • Figures 5A-5B depict the isolated nucleotide sequence and deduced amino acid sequence of the gapC gene for S. iniae (SEQ ID NO:19 and SEQ ID NO:20). In the figure, the asterisk represents a stop codon, and the underlined regions represent nucleotide sequences complementary to the primers used to isolate the genes from the bacterial chromosomes.
    • Figure 6 depicts the nucleotide sequence (SEQ ID NO:21) and deduced amino acid sequence (SEQ ID NO:22) of the GapC multiple epitope fusion protein.
    • Figures 7A-7E show a DNA alignment chart created by PileUp and displayed by Pretty software (a component of the GCG Wisconsin Package, version 10, provided by the SeqWeb sequence analysis package, version 1.1, of the Canadian Bioinformatics Resource). The figure depicts the isolated nucleotide sequences of the gapC genes from S dysgalactiae (DysGapC, Check 9344) (SEQ ID NO:11); S. agalactiae (AgalGapC. Check 2895) (SEQ ID NO:13); S. uberis (UberGapC, Check 5966) (SEQ ID NO:15); S. parauberis (PUberGapC, Check 9672) (SEQ ID NO:17); and S. iniae (IniaeGapC, Check 990) (SEQ ID NO:19). The previously known sequences of S. equisimilis (SeqGapC, Check 5841 ), S. pyogenes (SpyGapC, Check 4037), and a bovine GAPDH protein (BovGapC, check 5059) are also included. The length and weight parameters were the same for all sequences (1018 and 1.00, respectively). The parameters used in the DNA sequence comparison were as follows: Plurality--2.00; Threshold-1; AveWeight--I.00; AveMatch--1.00; AvMisMatch--0.00; Symbol comparison table-pileupdna.cmp; CompCheck--6876; GapWeight--5; GapLengthWeight--1; PileUp MSF--1018; Type--N; Check--3804. In the figure, dashes represent identical nucleotides; dots represent gaps introduced by the software used to generate the alignment chart, and tildes represent regions not included in the overall alignment due to differences in the length of the gene sequences.
    • Figures 8A-8C show an amino acid sequence alignment chart created by PileUp and displayed by Pretty (as above) that depicts the alignment of PolyGap4 (SEQ ID NO:22) with the deduced amino acid sequences of the native GapC proteins isolated from S. dysgalactiae DysGapC, Check 6731) (SEQ ID NO:12), S. agalactiae (AgalGapC, Check 1229) (SEQ ID NO:14), S. uberis (UberGapC, Check 8229) (SEQ ID NO:16), S. parauberis (PUberGapC, Check 8889) (SEQ ID NO:18), and S. iniae (IniaeGapC, check 8785) (SEQ ID NO:20). The previously known sequences of S. equisimilis (SeqGapC, Check 8252), S. pyogenes (SpyGapC, Check 6626) and a bovine GAPDH protein (BovGapC, Check 8479) are also included. In the figure, dashes represent identical amino acid residues; dots represent gaps introduced by the PileUp software, and tildes represent regions not included in the overall alignment due to differences in the length of the gene sequences.
    • Figure 9 shows a Kyte-Doolittle hydropathy plot, averaged over a window of 7, an Emini surface probability plot, a Karplus-Schulz chain flexibility plot, a Jameson-WoIf antigenic index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure plots for the GapC protein isolated from S. dysgal.
    • Figure 10 shows a Kyte-Doolittle hydropathy plot, averaged over a window of 7, an Emini surface probability plot, a Karplus-Schulz chain flexibility plot, a Jameson-Woif antigenic index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure plots for the GapC protein isolated from S. agal.
    • Figure 11 shows a Kyte-Doolittle hydropathy plot, averaged over a window of 7, an Emini surface probability plot, a Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure plots for the GapC protein isolated from S. uberis.
    • Figure 12 shows a Kyte-Doolittle hydropathy plot, averaged over a window of 7, an Emini surface probability plot, a Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure plots for the GapC protein isolated from S. parauberis.
    • Figure 13 shows a Kyte-Doolittle hydropathy plot, averaged over a window of 7, an Emini surface probability plot, a Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure plots for the GapC protein isolated from S. iniae.
    • Figure 14 shows a Kyte-Doolittle hydropathy plot, averaged over a window of 7, an Emini surface probability plot, a Karplus-Schulz chain flexibility plot, a Jameson-Wolf antigenic index plot, and both Chou-Fasman and Garnier-Osguthorpe-Robson secondary structure plots for LipoFGAP4 (SEQ ID NO:22), the chimeric GapC protein.
    • Figure 15 is a diagrammatic representation of the Chou-Fasman secondary structure plot for the GapC protein isolated from S. dysgal.
    • Figure 16 is a diagrammatic representation of the Chou-Fasman secondary structure plot for the GapC protein isolated from S. agal.
    • Figure 17 is a diagrammatic representation of the Chou-Fasman secondary structure plot for the GapC protein isolated from S. uberis.
    • Figure 18 is a diagrammatic representation of the Chou-Fasman secondary structure plot for the GapC protein isolated from S. parauberis.
    • Figure 19 is a diagrammatic representation of the Chou-Fasman secondary structure plot for the GapC protein isolated from and S. iniae.
    • Figure 20 is a diagrammatic representation of the Chou-Fasman secondary structure plot for LipoFGAP4 (SEQ ID NO:22), the chimeric GapC protein.
    • Figure 21 is a diagram of plasmid pPolyGap.1.
    • Figure 22 is a diagram of plasmid pPolyGap.2.
    • Figure 23 is a diagram of plasmid pPolyGap.3.
    • Figure 24 is a diagram of plasmid pPolyGap.4
    • Figure 25 is a diagram of plasmid polygap4.
    Detailed Description
  • The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., 1986, Blackwell Scientific Publications).
  • The following amino acid abbreviations are used throughout the text:
    • Alanine: Ala (A) Arginine: Arg (R)
    • Asparagine: Asn (N) Aspartic acid: Asp (D)
    • Cysteine: Cys (C) Glutamine: Gln (Q)
    • Glutamic acid: Glu (E) Glycine: Gly (G)
    • Histidine: His (H) Isoleucine: Ile (I)
    • Leucine: Leu (L) Lysine: Lys (K)
    • Methionine: Met (M) Phenylalanine: Phe (F)
    • Proline: Pro (P) Serine: Ser (S)
    • Threonine: Thr (T) Tryptophan: Trp (W)
    • Tyrosine: Tyr (Y) Valine: Val (V)
    A. Definitions
  • In this document, the following terms will be employed, and are intended to be defined as indicated below.
  • It must be noted that, as used in this document, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a Streptococcus GapC protein" includes a mixture of two or more such proteins, and the like.
  • The terms "GapC protein" and "GapC plasmin binding protein" (used interchangeably herein) or a nucleotide sequence encoding the same, intends a protein or a nucleotide sequence, respectively, which is derived from a GapC gene found in a variety of Streptococcus species, including, without limitation certain strains of group A streptococci (Lottenbery, R.,. et al., (1987) Infect. Immun. 55:1914-1918). The nucleotide sequence of representative Streptococcus gapC genes, and the corresponding amino acid sequence of the GapC proteins encoded by these genes, are depicted in the Figures. In particular, Figures 1 through 5 depict the isolated nucleotide sequences and isolated amino acid sequences of S. dysgalactiae (SEQ ID NO:11 and SEQ ID NO:12, respectively), S. agalactiae (SEQ ID NO:13 and SEQ ID NO:14, respectively), S. uberis (SEQ ID NO:15 and SEQ ID NO:16, respectively), S. parauberis (SEQ ID NO:17 and SEQ ID NO:18, respectively,), and S. iniae (SEQ ID NO:19 and SEQ ID NO:20, respectively). However, a GapC protein as defined herein is not limited to the depicted sequences as subtypes of each of these Streptococcus species are known and variations in GapC proteins will occur between them.
  • Representative gapC genes, derived from S. dysgalactiae, S. agalactiae, S. uberis, and S. parauberis, are found in the plasmids pET15bgapC, pMF521c, pMF521 a, pMF521d, and pMF521e, respectively.
  • Furthermore, the derived protein or nucleotide sequences need not be physically derived from the gene described above, but may be generated in any manner, including for example, chemical synthesis, isolation (e.g., from S. dysgalactiae) or by recombinant production, based on the information provided herein. Additionally, the term intends proteins having amino acid sequences substantially homologous (as defined below) to contiguous ammo acid sequences encoded by the genes, which display immunological and/or plasmin-binding activity.
  • Thus, the terms intend full-length, as well as immunogenic, truncated and partial sequences, and active analogs and precursor forms of the proteins. Also included in the term are nucleotide fragments of the gene that include at least about 8 contiguous base pairs, more preferably at least about 10-20 contiguous base pairs, and most preferably at least about 25 to 50, or more, contiguous base pairs of the gene, or any integers between these values. Such fragments are useful as probes and in diagnostic methods, discussed more fully below.
  • The terms also include those forms possessing, as well as lacking, a signal sequence, if such is present, as well as the nucleic acid sequences coding therefore. Additionally, the term intends forms of the GapC proteins which lack a membrane anchor region, and nucleic acid sequences encoding proteins with such deletions. Such deletions may be desirable in systems that do not provide for secretion of the protein. Furthermore, the plasmin-binding domains of the proteins, may or may not be present. Thus, for example, if the GapC plasmin-binding protein will be used to purify plasmin, the plasmin-binding domain will generally be retained. If the protein is to be used in vaccine compositions, immunogenic epitopes which may or may not include the plasmin-binding domain, will be present.
  • The terms also include proteins in neutral form or in the form of basic or acid addition salts depending on the mode of preparation. Such acid addition salts may involve free amino groups and basic salts may be formed with free carboxyls. Pharmaceutically acceptable basic and acid addition salts are discussed further below. In addition, the proteins may be modified by combination with other biological materials such as lipids (both those occurring naturally with the molecule or other lipids that do not destroy immunological activity) and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains, oxidation of sulfhydryl groups, glycosylation of amino acid residues, as well as other modifications of the encoded primary sequence.
  • The term therefore intends deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that do not substantially affect the immunogenicity and/or plasmin-binding affinity of the protein, are therefore within the definition of the reference polypeptide.
  • For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 or 20-50 conservative or non-conservative amino acid substitutions, or any integer between these values, so long as the desired function of the molecule remains intact.
  • In this regard, GapC proteins isolated from streptococci exhibit several variable regions in their amino acid sequences, located at amino acid positions 62 to 81; 102 to 112; 165 to 172; 248 to 271; and 286 to 305. These regions, which in S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis and S. iniae exhibit from 1 to 9 amino acid substitutions, are likely to be amenable to variation without substantially affecting immunogenic or enzymatic function.
  • Similarly, substitutions occurring in the transmembrane binding domain, if present, and the signal sequence, if present, normally will not affect immunogenicity. One of skill in the art may readily determine other regions of the molecule of interest that can tolerate change by reference to the protein structure plots shown in Figures 9 to 20 herein.
  • The term "streptococcal GapC protein" intends a GapC plasmin-binding protein, as defined above, derived from a streptococcal species that produces the same, including, but not limited to S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis, and S. iniae. For example, a "S. dysgalactiae GapC protein" is a GapC plasmin-binding protein as defined above, derived from S. dysgalactiae. Similarly, an "S. agalactiae GapC protein" intends a gapC binding protein derived from S. agalactiae. "Wild type" or "native" proteins or polypeptides refer to proteins or polypeptides isolated from the source in which the proteins naturally occur. "Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides are those prepared by chemical synthesis.
  • An "isolated" protein or polypeptide is a protein or polypeptide molecule separate and discrete from the whole organism with which the molecule is found in nature; or a protein or polypeptide devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
  • The term "functionally equivalent" intends that the amino acid sequence of a GapC plasmin-binding protein is one that will elicit a substantially equivalent or enhanced immunological response, as defined above, as compared to the response elicited by a GapC plasmin-binding protein having identity with the reference GapC plasmin-binding protein, or an immunogenic portion thereof.
  • The term "epitope" refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. Epitopes may include 3 to 5 amino acids, more preferably 5 to 10 amino acids, up to the full length of the reference molecule.
  • The term "multiple epitope" protein or polypeptide specifies a sequence of amino acids comprising an epitope as defined herein, which contains at least one epitope repeated two or more times within a linear molecule. The repeating sequence need not be directly connected to itself, is not repeated in nature in the same manner and, further, may be present within a larger sequence which includes other amino acids that are not repeated. For the purposes of this document, the epitope sequence may either be an exact copy of a wild-type epitope sequence, or a sequence which is "functionally equivalent" as defined herein refers to a multiple epitope protein or polypeptide as defined herein that is produced by recombinant or synthetic methods.
  • A "fusion" or "chimeric" protein or polypeptide is one in which amino acid sequences from more than one source are joined. Such molecules may be produced synthetically or recombinantly, as described further herein (see the section entitled "Production of GapC Plasmin-Binding Proteins" infra). Hence, the term "multiple epitope fusion protein or polypeptide" refers to a multiple epitope protein or polypeptide as defined herein which is made by either synthetic or recombinant means.
  • In this regard, a multiple epitope fusion protein comprising the variable regions in the amino acid sequences of the GapC proteins referred to above may be produced. The amino acid sequence for a representative GapC multiple epitope fusion protein, and a corresponding polynucleotide coding sequence, is depicted in Figures 6A-6C herein. Methods for recombinantly producing the protein, including a method for constructing the polyGap4 plasmid containing the chimeric coding sequence (diagramed in Figure 25) and a method for expressing the protein from the polyGap4 plasmid, are described in Examples 4 and 5 infra.
  • The terms "immunogenic" protein or polypeptide refer to an amino acid sequence which elicits an immunological response as described herein. An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the GapC plasmin-binding protein in question, with or without the signal sequence, membrane anchor domain and/or plasmin-binding domain, analogs thereof, or immunogenic fragments thereof. By "immunogenic fragment" is meant a fragment of a GapC plasmin-binding protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots. Figures 9 to 20 herein depict Kyte-Doolittle profiles for representative proteins.
  • Immunogenic fragments will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, and most preferably 25 or more amino acids, of the parent GapC plasmin-binding-binding protein molecule. There is no critical upper limit to the length of the fragment, which may comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes of GapC.
  • An "immunogenic composition" is a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest.
  • By "subunit vaccine composition" is meant a composition containing at least one immunogenic polypeptide, but not all antigens, derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of intact pathogen cells or particles, or the lysate of such cells or particles. Thus, a "subunit vaccine composition" is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof. A subunit vaccine composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from the pathogen.
  • By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • An "immunological response" to a composition or vaccine is the development in the host of a cellular and/ or antibody-mediated immune response to the composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance of the mammary gland to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host and/or a quicker recovery time.
  • By "nucleic acid immunization" is meant the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell, for the in vivo expression of an antigen, antigens, an epitope, or epitopes. The nucleic acid molecule can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal and mucosal administration, or the like, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule.
  • The term "treatment" as used herein refers to either (1) the prevention of infection or reinfection (prophylaxis), or (2) the reduction or elimination of symptoms of the disease of interest (therapy).
  • By "mastitis" is meant an inflammation of the mammary gland in mammals, including in cows, ewes, goats, sows, mares, and the like, caused by the presence of pathogenic microorganisms, such as S. uberis. The infection manifests itself by the infiltration of phagocytic cells in the gland. Generally, 4 clinical types of mastitis are recognized: (1) peracute, associated with swelling, heat, plain, and abnormal secretion in the gland and accompanied by fever and other signs of systemic disturbance, such as marked depression, rapid weak pulse, sunken eyes, weakness and complete anorexia; (2) acute, with changes in the gland similar to those above but where fever, anorexia and depression are slight to moderate; (3) subacute, where no systemic changes are displayed and the changes in the gland and its secretion are less marked: and (4) subclinical, where the inflammatory reaction is detectable only by standard tests for mastitis.
  • Standard tests for the detection of mastitis include but are not limited to, the California Mastitis Test, the Wisconsin Mastitis Test, the Nagase test, the electronic cell count and somatic cell counts used to detect a persistently high white blood cell content in milk. In general, a somatic cell count of about 300,000 to about 500,000 cells per ml or higher, in milk will indicate the presence of infection. Thus, a vaccine is considered effective in the treatment and/or prevention of mastitis when, for example, the somatic cell count in milk is retained below about 500,000 cells per ml. For a discussion of mastitis and the diagnosis thereof, see, e.g., The Merck Veterinary Manual: A Handbook of Diagnosis, Therapy, and Disease Prevention and Control for the Veterinarian, Merck and Co., Rahway, New Jersey, 1991.
  • By the terms "vertebrate," "subject," and "vertebrate subject" are meant any member of the subphylum Chordata, including, without limitation, mammals such as cattle, sheep, pigs, goats, horses, and humans; domestic animals such as dogs and cats; and birds, including domestic, wild and game birds such as cocks and hens including chickens, turkeys and other gallinaceous birds; and fish. The term does not denote a particular age. Thus, both adult and newborn animals, as well as fetuses, are intended to be covered.
  • A "nucleic acid" molecule can include, but is not limited to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
  • An "isolated" nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith. The term "isolated" in the context of a polynucleotide intends that the polynucleotide is isolated from the chromosome with which it is normally associated, and is isolated from the complete genomic sequence in which it normally occurs. "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • A "coding sequence" or a "nucleotide sequence encoding" a particular protein, is a nucleotide sequence which is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. A "complementary" sequence is one in which the nitrogenous base at a given nucleotide position is the complement of the nitrogenous base appearing at the same position in the reference sequence. To illustrate, the complement of adenosine is tyrosine, and vice versa; similarly, cytosine is complementary to guanine, and vice versa; hence, the complement of the reference sequence 5'-ATGCTGA-3' would be 5'-TACGACT-3'.
  • A "wild-type" or "native" sequence, as used herein, refers to polypeptide encoding sequences that are essentially as they are found in nature, e.g., the S. dysgalactiae GapC protein encoding sequences depicted in Figures 1A-1B (SEQ ID NO:12).
  • "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. "Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
  • "Homology" refers to the percent identity between two polynucleotide or two polypeptide moieties. Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank ± EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.
  • Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • By the term "degenerate variant" is intended a polynucleotide containing changes in the nucleic acid sequence thereof, that encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the degenerate variant is derived.
  • Techniques for determining amino acid sequence "similarity" are well known in the art. In general, "similarity" means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" then can be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a bacterial gene, the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria. Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A vector is capable of transferring gene sequences to target cells (e.g., bacterial plasmid vectors, viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • Typically, the terms "vector construct," "expression vector," "gene expression vector," "gene delivery vector," "gene transfer vector," and "expression cassette" all refer to an assembly which is capable of directing the expression of a sequence or gene of interest. Thus, the terms include cloning and expression vehicles, as well as viral vectors.
  • These assemblies include a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. The expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • DNA "control elements" refers collectively to transcription promoters, transcription enhancer elements, transcription termination sequences, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), translation termination sequences, upstream regulatory domains, ribosome binding sites and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. See e.g., McCaughan et al. (1995) PNAS USA 92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated. "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered "operably linked" to the coding sequence. Similarly, "control elements compatible with expression in a subject" are those which are capable of effecting the expression of the coding sequence in that subject.
  • A control element, such as a promoter, "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule.
  • A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.
  • As used herein, the terms 'label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH and α-β-galactosidase.
    • 2. MODES OF CARRYING OUT THE INVENTION
  • Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
  • Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
    • General Overview
  • We have discovered that the GapC protein is capable of eliciting an immune response in a vertebrate subject. Experiments performed in support have demonstrated that immunization of dairy cattle with the GapC protein of S. dysgalactiae conferred protection against experimental infection with this organism, and furthermore, conferred cross-protection against infection by S. uberis.
  • GapC is produced by a number of different streptococcus species. With the exception of several localized variable regions, the amino acid sequences of the GapC proteins produced by those strains are highly conserved. Therefore, it is desirable to construct multiple epitope GapC fusion proteins comprising antigenic determinants taken from both the highly conserved regions of GapC, and the unique regions of GapC proteins from several streptococcal species. Experiments performed in support have demonstrated that such a protein is capable of eliciting broad immunity against a variety of streptococcal infections while providing the additional economic advantage of minimizing the number of antigens present in the final formulation, and concomitantly reducing the cost of producing that formulation.
  • We describe GapC multiple epitope fusion proteins which have general structural formula (A)x--(B)y--(C)z representing a linear amino acid sequence. B is an amino acid sequence of at least five and not more than 1,000 amino acids of an antigenic determinant from a GapC protein, and y is an integer of 2 or more. A and C are each different from B, as well as being different from each other, and are independently an amino acid sequence of an antigenic determinant containing at least five and not more than 1,000 amino acids not immediately adjacent to B in nature. x and z are each independently an integer of 0 or more, wherein at least one of x and z is 1 or more.
  • Typically, A, B, and C are antigenic determinants from the GapC proteins of one or more bacterial species. In a preferred embodiment, A, B, and C are amino acid sequences comprising one or more antigenic determinants from the GapC protein of one or more of the following species of streptococcus: S. dysgalaetiae; S. agalactiae; S. uberis; S. parauberis, and S. iniae.
  • In this regard, Figures 9 through 13 show plots of the following for the streptococcal GapC proteins employed: Kyte-Doolittle hydrophathy, averaged over a window of 7; surface probability according to Emini; chain flexibility according to Karplus-Schulz; antigenicity index according to Jameson-Wolf; secondary structure according to Gamier-Osguthorpe-Robson; secondary structure according to Chou-Fasman; and predicted glycosylation sites. Figures 15 through 19 show plots of secondary structure according to Chou-Fasman for the aforementioned proteins. One of skill in the art can readily use the information presented in Figures 9 through 13 and 15 to 19 in view of the teachings of the present specification to identity antigenic regions which may be employed in constructing a chimeric protein.
  • Most preferably, A, B, and/or C include one or more variable regions of the GapC proteins from more than one streptococcus species. In this regard, Figures 8A-8C show an amino acid sequence alignment which illustrates regions of homology and variability that exist among GapC proteins from S dysgalactiae, S. agalactiae, S. uberis, S. parauberis, and S. iniae. Amino acid sequences for the GapC proteins of S. pyogenes and S. equisimilis, S. pyogenes are also included. In particular, several variable regions are located at amino acid positions 62 to 81; 102 to 112; 165 to 172; 248 to 271; and 286 to 305.
  • The multiple epitope fusion protein may also include spacer sequences interposed between A, B, and/or C. The spacer sequences are typically amino acid sequences of from 1 to 1,000 amino acids, may be the same or different as A, B, or C, and may be the same or different as each other.
  • The protein may also include a signal sequence and/or a transmembrane sequence. Examples of suitable signal sequences include the E. coli LipoF signal sequence, and the OmpF signal sequence. Examples of suitable transmembrane sequences include those associated with LipoF and OmpF.
  • We describe in detail the multiple epitope fusion protein Gap4. The amino acid sequence of Gap4 (SEQ ID NO:22), a multiple epitope GapC fusion protein, is shown in Figures 6A-6C, as is the polynucleotide sequence which encodes it (SEQ ID NO:21). Gap4 is a 47.905 kDa chimerie protein of 448 amino acids. Residues 1 to 27 are identical to amino acid residues 1 to 27 of the E. coli LipoF signal sequence. Residues 28 to 123 are identical to residues 1 to 96 of the S. dysgalactiae GapC protein. Residues 124 (leucine) and 125 (glutamic acid) are spacer amino acids. They are followed by residues 126 to 165, which are identical to residues 56 to 95 of S. parauberis as well as to the same residues of S. uberts. Residue 166 (isoleucine) is a spacer amino acid. Residues 167 to 208 are identical to residues 55 to 96 of the S. agalactiae GapC protein. Residues 209 (threonine) and 210 (serine) are spacer amino acids. Residues 211 to 448 are identical to residues 99 to 336 of the S. dysgalactiae GapC protein.
  • As expressed, Gap4 has a cysteine residue present at the amino terminal end of the mature protein. The LipoF signal sequence and cysteine residue are present to ensure that the chimeric molecule is efficiently secreted from the bacterial host cell and becomes bound to the host cell membrane via the lipid-moiety. The protein may then be extracted from the cell surface via differential solubilization with a detergent such as Sarkosyl or TritonX-100® (see Example 5 infra).
  • The GapC chimeric proteins or antigenic fragments thereof can be provided in subunit vaccine compositions. In addition to use in vaccine compositions, the proteins or antibodies thereto can be used as diagnostic reagents to detect the presence of infection in a vertebrate subject. Similarly, the genes encoding the proteins can be cloned and used to design probes to detect and isolate homologous genes in other bacterial swains. For example, fragments comprising at least about 15-20 nucleotides, more preferably at least about 20-50 nucleotides, and most preferably about 60-100 nucleotides, or any integer between these values, will find use in these embodiments.
  • The vaccine compositions can be used to treat or prevent a wide variety of bacterial infections in vertebrate subjects. For example, vaccine compositions including GapC multiple epitope fusion proteins comprising antigenic determinants from S. dysgalactiae. S. uberis, S. parauberis, S. iniae, and/or group B streptococci (GBS) such as S. agalactiae, can be used to treat streptococcal infections in vertebrate subjects that are caused by these or other species. In particular, S uberis and S. agalactiae are common bacterial pathogens associated with mastitis in bovine, equine, ovine and goat species. Additionally, group B streptococci, such as S. agalactiae, are known to cause numerous other infections in vertebrates, including septicemia, meningitis, bacteremia, impetigo, arthritis, urinary tract infections, abscesses, spontaneous abortion etc. Hence, vaccine compositions containing chimeric GapC proteins will find use in treating and/or preventing a wide variety of streptococcal infections.
  • Similarly, GapC multiple epitope fusion proteins comprising antigenic determinants derived from other bacterial genera such as Staphylococcus, Mycobacterium, Escherichia, Pseudomonas, Nocardia, Pasteurella, Clostridium and Mycoplasma will find use for treating bacterial infections caused by species belonging to those genera. Thus, it is readily apparent that chimeric GapC proteins can be used to treat and/or prevent a wide variety of bacterial infections in numerous species.
  • The GapC multiple epitope fusion proteins can be used in vaccine compositions either alone or in combination with other bacterial, fungal, viral or protozoal antigens. These other antigens can be provided separately or even as fusion proteins comprising the GapC chimeric protein fused to one or more of these antigens. For example, other immunogenic proteins from S. uberis, such as the CAMP factor, hyaluronic acid capsule, hyaluronidase, R-like protein and plasminogen activator, can be administered with the chimeric GapC protein. Additionally, immunogenic proteins from other organisms involved in mastitis, such as from the genera Staphylococcus, Corynebacterium, Pseudomonas, Nocardia, Clostridium, Mycobacteriurn, Mycoplasma, Pasteurella, Prototheca, other streptococci, coliform bacteria, as well as yeast, can be administered along with the GapC fusion proteins described herein to provide a broad spectrum of protection. Thus, for example, immunogenic proteins from Staphylococcus aureus, Str. agalactiae, Str. dysgalactiae, Str. zooepidemicus, Corynebacterium pyogenes, Pseudomonas aeruginosa, Nocardia asteroides, Clostridium perfringens, Escherichia coli, Enterobacter aerogenes and Klebsiella spp. can be provided along with the GapC plasmin-binding proteins.
    • Production of GapC Multiple Epitope Fusion Proteins
  • The above-described chimeric proteins and active fragments and analogs derived from the same, can be produced by recombinant methods as described herein. These recombinant products can take the form of partial protein sequences, full-length sequences, precursor forms that include signal sequences, or mature forms without signals.
  • The GapC plasmin-binding protein DNA sequences used to construct the chimeric proteins can be isolated by a variety of methods known to those of skill in the art. See, e.g., Sambrook et al., supra. Methods for isolating, cloning and sequencing the gene sequences encoding GapC proteins from S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis, and S. iniae are detailed in Examples 1,2 and 3, infra.
  • After isolating and cloning the desired GapC protein sequences, polynucleotide sequences encoding the chimeric proteins are constructed using standard recombinant techniques including PCR amplification, restriction endonuclease digestion and ligation. See, e.g., Sambrook et al., supra. Methods for constructing Gap4 are detailed in Example 4, infra.
  • Alternatively, the DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned. The DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.
  • Once coding sequences for the desired proteins have been prepared, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage λ (E. coil), pBR322 (E. coli), pACYC177 (E. coil), pKT230 (gram-negative bacteria), pGV1106 (grain-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-(E. coli gram-negative bacteria), pHV14 ((E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, Sambrook et al, supra; DNA Cloning, supra; B. Perbal, supra.
  • The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. If a signal sequence is included, it can either be the native, homologous sequence, or a heterologous sequence. For example, the LipoF signal sequence is added to the amino-terminal region of the chimeric protein Gap4 to permit secretion of the protein after expression. See Examples 4E and 5, infra. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739 ; 4,425,437 ; 4,338,397 .
  • Other regulatory sequences which allow for regulation of expression of the protein sequences relative to the growth of the host cell may also be desirable. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. It may also be desirable to produce mutants or analogs of the GapC plasmin-binding protein. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al., supra; DNA cloning, supra; Nucleic Acid Hybridization, supra.
  • The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ('MDBK") cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis and Streptococcus spp., will find use with the present expression constructs. Useful yeast hosts include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polyrnorpha, Kluyveromyces friagilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
  • Depending on the expression system and host selected, the proteins may be produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into the growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • The proteins may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.
  • The chimeric GapC plasmin-binding proteins, or their fragments, can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffmity chromatography, using known procedures.
  • Monoclonal antibodies to the chimeric GapC plasmin-binding proteins and to the fragments thereof, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see also U.S. Patent Nos. 4,341,761 ; 4,399,121 ; 4,427,783 ; 4,444,887 ; 4,452,570 ; 4,466,917 ; 4,472,500 , 4,491,632 ; and 4,493,890 . Panels of monoclonal antibodies produced against the chimeric GapC plasmin-binding proteins, or fragments thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes.
    • Vaccine Formulations and Administration
  • The GapC multiple epitope fusion proteins can be formulated into vaccine compositions, either alone or in combination with other antigens, for use in immunizing subjects as described below. Methods of preparing such formulations are described in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18 Edition, 1990. Typically, the vaccines are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The active immunogenic ingredient is generally mixed with a compatible pharmaceutical vehicle, such as, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof, In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.
  • Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation. Adjuvants may include for example, muramyl dipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art.
  • The chimeric GapC plasmin-binding protein may be linked to a carrier in order to increase the immunogenicity thereof. Suitable carriers include large, slowly metabolized macromolecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.
  • The chimeric GapC plasmin-binding proteins may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.
  • Other suitable carriers for the chimeric GapC plasmin-binding proteins include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S. Patent No. 5,071,651 . Also useful is a fusion product of a viral protein and the subject chimeric proteins made by methods disclosed in U.S. Patent No. 4,722,840 . Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the proteins may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.
  • Furthermore, the chimeric GapC plasmin-binding proteins (or complexes thereof) may be formulated into vaccine compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an immune response in a subject to which the composition is administered. In the treatment and prevention of mastitis, for example, a "therapeutically effective amount" would preferably be an amount that enhance resistance of the mammary gland to new infection and/or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered somatic cell count in milk from the infected quarter. For example, the ability of the composition to retain or bring the somatic cell count (SCC) in milk below about 500,000 cells per ml, the threshold value set by the International Dairy Federation, above which, animals are considered to have clinical mastitis, will be indicative of a therapeutic effect.
  • The exact amount is readily determined by one skilled in the art using standard tests. The chimeric GapC plasmin-binding protein concentration will typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. With the present vaccine formulations, 5 to 500 µg of active ingredient per ml of injected solution should be adequate to raise an immunological response when a dose of 1 to 3 ml per animal is administered.
  • To immunize a subject, the vaccine is generally administered parenterally, usually by intramuscular injection. Other modes of administration, however, such as subcutaneous, intraperitoneal and intravenous injection, are also acceptable. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. The subject is immunized by administration of the vaccine in at least one dose, and preferably two doses. Moreover, the animal may be administered as many doses as is required to maintain a state of immunity to infection.
  • Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations. For suppositories, the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • Controlled or sustained release formulations are made by incorporating the protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. The chimeric GapC plasmin-binding proteins can also be delivered using implanted mini-pumps, well known in the art.
  • The chimeric GapC plasmin-binding proteins can also be administered via a carrier virus which expresses the same. Carrier viruses which will find use include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus. By way of example, vaccinia virus recombinants expressing the novel proteins can be constructed as follows. The DNA encoding the particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome. The resulting TK recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
  • An alternative route of administration involves gene therapy or nucleic acid immunization. Thus, nucleotide sequences (and accompanying regulatory elements) encoding the subject chimeric GapC plasmin-binding proteins can be administered directly to a subject for in vivo translation thereof. Alternatively, gene transfer can be accomplished by transfecting the subjects cells or tissues ex vivo and reintroducing the transformed material into the host. DNA can be directly introduced into the host organism, i.e., by injection (see International Publication No. WO/90/11092 ; and Wolff et al. (1990) Science 247:1465-1468). Liposome-mediated gene transfer can also be accomplished using known methods. See, e.g., Hazinski. et al. (1991) Am. J. Respir. Cell Mol. Biol. 4:206-209, Brigham et al. (1989) Am. J. Med Sci. 298:278-281; Canonico et al. (1991) Clin. Res. 39:219A; and Nabel et al. (1990) Science 249:1285-1288. Targeting agents, such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to infection.
    • Diagnostic Assays
  • As explained above, the chimeric GapC plasmin-binding proteins may also be used as diagnostics to detect the presence of reactive antibodies of streptococcus, for example S. dysgalactiae, in a biological sample in order to determine the presence of streptococcus infection. For example, the presence of antibodies reactive with chimeric GapC plasmin-binding proteins can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • The aforementioned assays generally involve separation of unbound antibody in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • Typically, a solid support is first reacted with a solid phase component (e.g., one or more chimeric GapC plasmin-binding proteins) under suitable binding conditions such that the component is sufficiently immobilized to the support. Sometimes, immobilization of the antigen to the support can be enhanced by first coupling the antigen to a protein with better binding properties. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules to the antigens, are well known to those of ordinary skill in the art. See, e.g., Brinkley, M.A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl. Biochem. (1984) 6:56-63 and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30:117-124.
  • After reacting the solid support with the solid phase component, any non-immobilized solid-phase components are removed from the support by washing, and the support-bound component is then contacted with a biological sample suspected of containing ligand moieties (e.g., antibodies toward the immobilized antigens) under suitable binding conditions. After washing to remove any non-bound ligand, a secondary binder moiety is added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound ligand. The presence of the secondary binder can then be detected using techniques well known in the art.
  • More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a chimeric GapC plasmin-binding protein. A biological sample containing or suspected of containing anti-chimeric GapC plasmin-binding protein immunoglobulin molecules is then added to the coated wells. After a period of incubation sufficient to allow antibody binding to the immobilized antigen, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample antibodies, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • Thus, in one particular embodiment, the presence of bound anti-chimeric GapC plasmin-binding antigen ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands. A number of anti-bovine immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art. An appropriate enzyme substrate is then used to generate a detectable signal. In other related embodiments, competitive-type ELISA techniques can be practiced using methods known to those skilled in the art.
  • Assays can also be conducted in solution, such that the chimeric GapC plasmin-binding proteins and antibodies specific for those proteins form complexes under precipitating conditions. In one particular embodiment, chimeric GapC plasmin-binding proteins can be attached to a solid phase particle (e.g., an agarose bead or the like) using coupling techniques known in the art, such as by direct chemical or indirect coupling. The antigen-coated particle is then contacted under suitable binding conditions with a biological sample suspected of containing antibodies for the chimeric GapC plasmin-binding proteins. Cross-linking between bound antibodies causes the formation of particle-antigen-antibody complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation. The reaction mixture can be analyzed to determine the presence or absence of antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.
  • In yet a further embodiment, an immunoaffinity matrix can be provided, wherein a polyclonal population of antibodies from a biological sample suspected of containing anti-chimeric GapC plasmin-binding molecules is immobilized to a substrate. In this regard, an initial affinity purification of the sample can be carried out using immobilized antigens. The resultant sample preparation will thus only contain anti-streptococcus moieties, avoiding potential nonspecific binding properties in the affinity support. A number of methods of immobilizing immunoglobulins (either intact or in specific fragments) at high yield and good retention of antigen binding activity are known in the art. Not being limited by any particular method, immobilized protein A or protein G can be used to immobilize immunoglobulins.
  • Accordingly, once the immunoglobulin molecules have been immobilized to provide an immunoaffinity matrix, labeled chimeric GapC plasmin-binding proteins are contacted with the bound antibodies under suitable binding conditions. After any non-specifically bound antigen has been washed from the immunoaffinity support, the presence of bound antigen can be determined by assaying for label using methods known in the art.
  • Additionally, antibodies raised to the chimeric GapC plasmin-binding proteins, rather than the chimeric GapC plasmin-binding proteins themselves, can be used in the above-described assays in order to detect the presence of antibodies to the proteins in a given sample. These assays are performed essentially as described above and are well known to those of skill in the art.
  • The above-described assay reagents, including the chimeric GapC plasmin-binding proteins, or antibodies thereto, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. The kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.
    • Deposits of Strains
  • A deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, under the provisions of the Budapest Treaty. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. The designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.
  • Should there be a discrepancy between the sequence presented in the present application and the sequence of the gene of interest in the deposited plasmid due to routine sequencing errors, the sequence in the deposited plasmid controls.
    Bacterial Strain Plasmid Deposit Date ATCC No.
    XLI Blue MRF pPolyGap.1 May 31, 2000 PTA-1981
    XLI Blue MRF pPolyGap.2 May 31, 2000 PTA-1974
    XLI Blue MRF pPolyGap.3 May 31, 2000 PTA-1979
    XLI Blue MRF pPolyGap.4 May 31, 2000 PTA-1980
    XLI Blue MRF polygap4 May 31, 2000 PTA-1978
  • Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
    • C. Experimental EXAMPLE 1 Preparation of Chromosomal DNA
  • A clinical S. dysgalactiae isolate from a case of bovine mastitis (ATCC Accession No. ATCC43078) was obtained from the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209), and was used as a source of DNA. The organism was routinely grown on TSA sheep blood agar plates (PML Microbiologicals, Mississauga, Ontario) at 37° C for 18 hours, or in Todd-Hewitt broth (Oxoid Ltd., Hampshire, England) supplemented with 0.3% yeast extract (THB-YE) at 37° C, 5% CO2.
  • Chromosomal DNA was prepared from S. dysgalactiae grown in 100 ml of THB-YE supplemented with 20 mM glycine for approximately 6 hours, until an A600 of 0.8 to 1.0 was reached. Cells were harvested and re-suspended in 50 mM EDTA, 50 mM Tris-HCl, 0.5% Tween-20® (Sigma, St. Louis, MO) and supplemented with RNase A (200 mg/ml), proteinase K (20 mg/ml), lysozyme (100 mg/ml) and mutanolysin (100 mg/ml). (all enzymes purchased from SIGMA, St. Louis, MO). Following bacterial lysis for 30 minutes at 37° C with vigorous shaking, guanidine hydrochloride and Twee-2®, pH 5.5, were mixed with the lysate to give a final concentration of 0.8 M and 5%, respectively. This mixture was incubated at 50° C for 30 minutes. The chromosomal DNA was then purified using a Qiagen genomic-tip 100g (Qiagen, Santa Clarita, California) and precipitated using 0.7 volumes of isopropanol. The resulting pellet was washed in 70% ethanol and re-suspended in 0.5 ml 10 mM Tris-HCl, pH 8.8.
  • Chromosomal DNA from S. agalactiae, S. uberis and, S. parauberis was isolated essentially as described above, from strains designated ATCC 27541, 9927, and 13386, respectively. Chromosomal DNA from S. iniae was also isolated as above from a strain designated 9117 obtained from Mount Sinai Hospital, University of Toronto, Canada.
    • EXAMPLE 2 Amplification and Cloning of gapC genes from S. dysgalactiae, S. uberis, S. parauberis, S. agalactiae and S. iniae.
  • The polynucleotide sequences encoding GapC from S. dysgalactiae, S. uberis, S. parauberis, S. agalactiae and S. iniae were initially isolated from chromosomal DNA by PCR amplification. The primers used to PCR-amplify the gapC genes from all species were gapC1 (SEQ ID NO:1) and gapC1r (SEQ ID NO:2), shown in Table 1. In the table, underlining denotes nucleotides added to the original sequences (i.e., nucleotides added to the 5' end of the original sense strand sequence and to the 3' end of the original anti-sense strand sequence, respectively, of the gapC coding region being amplified ), and bolding indicates the location of restriction endonuclease recognition sites.
  • PCR was carried out using Vent DNA polymerase (New England Biolabs, Mississauga, ON, Canada). A reaction mixture containing 0.2 µg of genomic DNA, 1pM of each of the preceding primers, 100 pM each of dATP, dTTP, dCTP and dGTP, 10mM Tris HCL, pH9; 1.5mM MgCl2 , 50mM HCL, 1.5 units Taq DNA polymerase (Pharmacia, Quebec, Canada) was incubated for 40 amplification cycles of 40 seconds at 94°C, 40 seconds at 55 °C, and 1 minute, 20 seconds at 72°C, and then for a single cycle of 10 minutes at 72°C.
  • The resulting PCR reaction products were then digested with NdeI and BamHI. In the case of the S. dysgalactiae gapC product, the fragment was cloned directly into the same sites of pET15b (Novagen, Madison, Wisconsin) after the plasmid was digested with the same enzymes. The resulting construct was denominated pET15bgapC. In the case of the S. agalactiae, S. uberis, S. parauberis and S. iniae sequences, each was first cloned into pPCR-Script using the cloning protocol described in the PCR-Script Amp Cloning Kit (Stratagene, La Jolla, California), subsequently excised using NdeI and BamHI, and finally re-cloned into the corresponding sites of pET15b using conventional cloning protocols (see e.g., Sambrook et al., supra).
  • The plasmids containing the S. agalactiae, S. uberis, S. parauberis and S. iniae sequences were designated pMF521c-inv, pMF521a-inv, pMF521d-inv, and pMF521e-inv, respectively. Table 1: Sequence Identification Numbers and Corresponding Nucleotide and Amino Acid Sequences
    SEQ ID NO. Name Nucleotide Sequence (5' to 3')
    gapCl
    Figure imgb0001
    gapClr
    Figure imgb0002
    Gap-1 AAA AAA GGA TCC GGT ATG GTA GTT AAA GTT GG
    Gap-2
    Figure imgb0003
    Gap-3 AAA AAA CTC GAG GGT ACT GTA GAA GTT AAA G
    Gap-4
    Figure imgb0004
    Gap-5
    Figure imgb0005
    Gap-6
    Figure imgb0006
    Gap-7
    Figure imgb0007
    Gap-8
    Figure imgb0008
    Streptococcus dysgalactiae gapC gene (see Figure 1)
    Streptococcus dysgalactiae GapC protein
    Streptococcus agalactiae gapC gene (see Figure 2)
    Streptococcus agalactiae GapC protein
    Streptococcus uberis gapC gene (see Figure 3)
    Streptococcus uberis GapC protein
    Streptococcus parauberis gapC gene (see Figure 4)
    Streptococcus parauberis GapC protein
    Streptococcus iniae gapc gene (see Figure 5)
    Streptococcus iniae GapC protein
    Gap4 chimeric gapC gene (see Figure 6)
    Gap4 chimeric GapC protein
    • EXAMPLE 3 Sequencing of gapC genes
  • The genes isolated and cloned in the preceding examples were sequenced using fluorescence tag terminators on an ABI 373 DNA automatic sequencer (Applied Biosystems, Emeryville, California) at the Plant Biotechnology Institute (PBI, Saskatoon, Saskatchewan, Canada).
  • The nucleotide sequences so determined, and the corresponding amino acid sequences deduced therefrom, are shown in Figures 1 through 5.
    • EXAMPLE 4 Construction of a Chimeric gapC Gene
  • A chimeric gapC gene composed of sequences from S. dysgalactiae, S. parauberis, and S. agalactiae was constructed in a three-step process using pAA556, a standard tac-inducible expression plasmid derived from the plasmid pGH432 that contains the signal sequence from the E. Coli ompF gene.
  • The partial gapC gene sequences used to construct the chimeric gene were prepared by PCR amplification of selected polynucleotide sequences from the genomic gapC genes isolated above, using the primers Gap-1 through Gap-8. The primer sequences are depicted in Table 1.
  • After assembly, the chimeric gene, sans the ompF signal sequence, was then excised from pAA556 and inserted into the plasmid pAA555, a pGH432 derivative that is a standard tac-inducible expression plasmid containing the signal sequence from the E. coli ompF gene.
    • A. Construction of pPolyGap.1
  • In the first step, the first 288 bases of the S. dysgalactiae gapC gene were PCR amplified using the primers Gap-1 and Gap-2.
  • PCR amplification was carried out as follows: 1.6 µg of template DNA was combined in a reaction mixture containing 20 pM each of primer Gap-1 (SEQ ID NO:1) and Gap-2 (SEQ ID NO:2), 200 µm each of dATP, dCTP, dGTP and dTTP, 2.5mM MgSO4, PCR Buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), and 1 unit Taq DNA polymerase (Pharmacia, Quebec, Canada). The mix was amplified for 1 cycle of 1 minute at 95°C, then for 29 cycles of 1 minute at 95°C, 1 minute at 55°C, and 30 seconds at 72°C, and finally for 1 cycle of 10 minutes at 4°C.
  • The amplification product was then digested with BamHI and NcoI and inserted into the same sites of an pAA556 vector. The resulting plasmid construct, designated pPolyGap.1, is illustrated in Figure 21.
    • B. Construction of pPolyGap.2
  • A PCR product representing bases 170-285 of the S. parauberis gapC gene was then obtained using the primers Gap-3 (SEQ ID NO:5) and Gap-4 (SEQ ID NO:6). This product codes for an amino acid sequence identical to the corresponding amino acid sequence found in the S. uberis gapC gene. PCR amplification was carried out essentially as above, except using 2 µg of template DNA.
  • The S. parauberis PCR product and the pPolyGap1plasmid were both digested with Xho1 and Nco1, and the PCR product was ligated into the corresponding sites in the vector. This construct, called pPolyGap.2, is illustrated in Figure 22.
    • C. Construction of pPolyGap.3
  • Nucleotides 166-288 of the S. agalactiae gapC gene were amplified using PCR primers Gap-5 (SEQ ID NO:7) and Gap-6 (SEQ ID NO:8) as in Example 4B above.
  • The PCR product obtained was digested with ClaI and NcoI, then inserted into the same sites of pPolyGap2 immediately downstream of the S. parauberis sequence. pPolyGap3 is diagramed in Figure 23.
    • D. Construction of pPolyGap.4
  • The final step in constructing the chimeric gene involved the insertion of the remaining S. dysgalactiae gapC sequence (nucleotides 295-1011) in-frame and immediately downstream of the S. agalactiae sequence.
  • The S. dysgalactiae sequence was first PCR amplified using the primers Gap-7 (SEQ ID NO:9) and Gap-8 (SEQ ID NO:10) as in Example 4A above. The amplification product was then digested with the enzyme GamHi/NcoI, as was the pPolyGap.3 vector, and the fragment was then ligated into the corresponding vector sites.
  • This final step resulted in the plasmid pPolyGap.4 containing the complete gapC chimeric gene construct comprising an S. dysgalactiae gapC backbone with unique sequences from S. parauberis as well as S. agalactiae. See Figure 24.
    • E. Cloning of the Chimeric gapC Gene into pAA55: Construction of PolyGap.4
  • The chimeric gapc gene constructed in the preceding steps was excised from pAA556 by digestion with BamH1 and NcoI and inserted into the plasmid pAA555 digested with the same enzymes. pAA555 is identical to pAA556 except that the former plasmid contains the LipoF signal sequence, and provides for the addition of a cysteine at the amino terminal end of the mature GapC protein. The N-terminal cysteine was added to insure the chimeric protein's efficient secretion of from the cell and binding to the membrane via the lipid-moiety. The coding sequence of the PolyGap4 plasmid construct is shown in Figure 25.
    • EXAMPLE 5 Expression and Isolation of the Chimeric GapC protein
  • PolyGap4 is used to transform E. coli J5 in the presence of polyethlene glycol (Kurien and Scofield (1995) BioTeclaniques 18:1023-1026).
  • The transformed cells carrying pPolyGap4 are grown to logarithmic phase in LB media at 37°C with shaking. Expression of the chimeric GapC protein is then induced by adding IPTG to a final concentration of 1mM and incubating the cells at 37°C for an additional 4 hours.
  • The chimeric GapC protein is then extracted from the cell surface by differential solubilization. The cells are collected by centrifugation and re-suspended in a volume of resuspension buffer (0.85% NaCl solution containing 0.6% sarkosyl) equal to 1/10th the original culture volume. The suspension is incubated at room temperature for 30 minutes with gentle shaking. The cells are collected by cenfrifuation and the supernatant containing the chimeric GapC protein is passed through a 0.2 µm membrane filter. Aliquots of the sterile supernatant are analyzed by SDS-PAGE and Western blots using a rabbit anti-GapC polyclonal antibody.
    • EXAMPLE 6 Immunization of Animals with the Chimeric GapC protein
  • Vaccines were formulated in such a fashion that they contained 100 µg/ml of purified chimeric GapC protein in the oil-based adjuvant VSA3 (VIDO, Saskatoon, Saskatchewan, Canada). VSA3 is a combination of Emulsigen Plus™ (MVP Laboratories, Ralston, Nebraska) and dimethyldioctadecyl ammonium bromide (Kodak, Rochester, New York).
  • Non-lactating Holstein cows with no history of S. dysgalactiae infection are obtained. Two weeks prior to vaccination, all animals are treated with 300 mg of Cephapirin per quarter (Cepha-dry™, Ayerst Laboratories, Montreal, Canada), in order to resolve any pre-existing udder infection prior to the vaccination step.
  • Groups of experimental animals are immunized subcutaneously with two doses of vaccines containing the chimeric GapC protein or a placebo with a three-week interval between immunizations. Ten days to two weeks following the second immunization, animals are exposed to 500-1,000 colony forming units of S. dysgalactiae delivered into three quarters with an udder infusion cannula. The fourth quarter on each animal serves as an un-infective control.
  • All animals are examined daily for clinical signs of disease and samples from all udder quarters are collected on each day. Samples are observed for consistency and antibody titre, somatic cell counts, and bacterial numbers are determined.
    • EXAMPLE 7 Determination of Antibodies Specific for the Chimeric GapC protein
  • GapC-specific antibodies in bovine serum are measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre plates (NUNC, Naperville, Illinois) are coated by adding 0.1 microgram per well purified chimeric protein in 50mM sodium carbonate buffer, pH 9.6, incubated overnight at 4° C. The liquid is removed and the wells are blocked with 3% bovine serum albumin for 1 hr at 37° C. Serial dilutions of bovine serum (from 1:4 to 1:64,000) are added to the wells and incubated for 2 hours at room temperature. The wells are aspirated, washed and incubated with 100 µl of alkaline phosphatase-conjugated goat anti-bovine IgG (Kirkgaard & Perry Laboratories Inc., Gaithersburg, Maryland) for 1 hr at room temperature. The wells are washed again, and 100 µl of p-nitrophenol phosphate (Sigma, St. Louis, Missouri) is added as a substrate to detect alkaline phosphatase activity. The absorbance at 405 nanometers is recorded following 1 hr incubation with the substrate at room temperature.
    • EXAMPLE 8 Bacterial Colonization
  • Bacteria are enumerated by spreading serial dilutions (100 to 10-6) directly onto TSA sheep blood agar plates followed by overnight incubation at 37°C, 5% CO2. Colonization is defined as >500 cfu/ml of the challenge organism recovered.
  • To confirm that the bacteria recovered from milk secretions are S. dysgalactiae, selected colonies recovered from each animal are tested using an API strep-20 test (bioMerieux SA, Hazelwood, Missouri) according to the manufacturer's instructions. This test identifies Streptococcus species according to an analytical profile compiled on the basis of enzymatic activity and sugar fermentation, using either an analytical profile index or identification software.
  • The relationship between anti-GapC titer and bacterial colonization is also determined.
    • EXAMPLE 9 Detemination of inflammatory response
  • Inflammatory response is measured as a function of mammary gland somatic cell count i.e., lymphocytes, neutrophils, and monocytes). Somatic cell counts are measured using standard techniques recommended by Agriculture and AgriFood Canada (IDF50B (1985): Milk and Milk Products--Methods of Sampling in a Coulter counter). Samples are read within 48 hours of collection and fixation, at days 1 through 7 post challenge.
  • The numbers of somatic cells present in the gland are determined on each day post challenge.
    • SEQUENCE LISTING
    • <110> University of Saskatchewan
      Potter, Andrew A.
      Perez-Casal, José
      Fontaine, Michael
    • <120> IMMUNIZATION OF DAIRY CATTLE WITH CHIMERIC GAPC PROTEIN AGAINST STREPTOCOCCUS INFECTION
    • <130> O8-891816WO
    • <140>
      <141>
    • <160> 22
    • <170> PatentIn Ver. 2.0
    • <210> 1
      <211> 37
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer gapC1
    • <400> 1
      ggcggcggca tatggtagtt aaagttggta ttaacgg    37
    • <210> 2
      <211> 35
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer gapC1r
    • <400> 2
      gcggatcctt atttagcgat ttttgcaaag tactc    35
    • <210> 3
      <211> 32
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer gap-1
    • <400> 3
      aaaaaaggat ccggtatggt agttaaagtt gg    32
    • <210> 4
      <211> 39
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-2
    • <400> 4
      aaaaaaccat ggttactcga gtgcttccag aacgatttc    39
    • <210> 5
      <211> 31
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-3
    • <400> 5
      aaaaaactcg agggtactgt agaagttaaa g    31
    • <210> 6
      <211> 45
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-4
    • <400> 6
      aaaaaaccat ggttaatcga tttcaagaac gatttcaaca ccgtc    45
    • <210> 7
      <211> 34
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-5
    • <400> 7
      aaaaaaatcg atggtactgt tgaagttaaa gaag    34
    • <210> 8
      <211> 45
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-6
    • <400> 8
      aaaaaaccat ggttaactag ttgcttcaag aacgatttct acgcc    45
    • <210> 9
      <211> 35
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-7
    • <400> 9
      aaaaaaacta gtttctttgc taaaaaagaa gctgc    35
    • <210> 10
      <211> 42
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap-8
    • <400> 10
      aaaaaaccat ggctattatt tagcgatttt tgcaaaatac tc    42
    • <210> 11
      <211> 1011
      <212> DNA
      <213> Streptococcus dysgalactiae
    • <220>
      <221> CDS
      <222> (1)..(1011)
    • <400> 11
      Figure imgb0009
      Figure imgb0010
      Figure imgb0011
    • <210> 12
      <211> 336
      <212> PRT
      <213> Streptococcus dysgalactiae
    • <400> 12
      Figure imgb0012
      Figure imgb0013
    • <210> 13
      <211> 1011
      <212> DNA
      <213> Streptococcus agalactiae
    • <220>
      <221> CDS
      <222> (1)..(1011)
    • <400> 13
      Figure imgb0014
      Figure imgb0015
      Figure imgb0016
    • <210> 14
      <211> 336
      <212> PRT
      <213> Streptococcus agalactiae
    • <400> 14
      Figure imgb0017
      Figure imgb0018
    • <210> 15
      <211> 1011
      <212> DNA
      <213> Streptococcus uberis
    • <220>
      <221> CDS
      <222> (1)..(1011)
    • <400> 15
      Figure imgb0019
      Figure imgb0020
      Figure imgb0021
    • <210> 16
      <211> 336
      <212> PRT
      <213> Streptococcus uberis
    • <400> 16
      Figure imgb0022
      Figure imgb0023
    • <210> 17
      <211> 1011
      <212> DNA
      <213> Streptococcus parauberis
    • <220>
      <221> CDS
      <222> (1)..(1011)
    • <400> 17
      Figure imgb0024
      Figure imgb0025
      Figure imgb0026
    • <210> 18
      <211> 336
      <212> PRT
      <213> Streptococcus parauberis
    • <400> 18
      Figure imgb0027
      Figure imgb0028
    • <210> 19
      <211> 1011
      <212> DNA
      <213> Streptococcus iniae
    • <220>
      <221> CDS
      <222> (1)..(1011)
    • <400> 19
      Figure imgb0029
      Figure imgb0030
      Figure imgb0031
    • <210> 20
      <211> 336
      <212> PRT
      <213> Streptococcus iniae
    • <400> 20
      Figure imgb0032
      Figure imgb0033
    • <210> 21
      <211> 1347
      <212> DNA
      <213> Artificial Sequence
    • <220>
      <223> Description of Artificial Sequence: primer Gap4 chimeric GapC protein
    • <220>
      <221> CDS
      <222> (1)..(1347)
    • <400> 21
      Figure imgb0034
      Figure imgb0035
      Figure imgb0036
    • <210> 22
      <211> 448
      <212> PRT
      <213> Artificial Sequence
    • <400> 22
      Figure imgb0037
      Figure imgb0038
      Figure imgb0039

Claims (19)

  1. A multiple epitope fusion polypeptide comprising an amino acid sequence or a sequence with at least 80% sequence identity thereto, the amino acid sequence comprising the sequence shown as Figure 6 (SEQ ID NO: 22) but lacking an E. coli LipoF signal sequence depicted as residues 1 to 27 of Figure 6 (SEQ ID NO: 22).
  2. A multiple epitope fusion polypeptide comprising an amino acid sequence according to Claim 1 together with a signal sequence.
  3. A multiple epitope fusion polypeptide according to Claim 2, in which the signal sequence comprises the amino acid sequence depicted at positions 1-27 of Figure 6 (SEQ ID NO: 22).
  4. A multiple epitope fusion polypeptide according to Claim 2 or 3, in which the multiple epitope fusion polypeptide comprises the amino acid sequence depicted in Figure 6 (SEQ ID NO: 22).
  5. A polynucleotide sequence encoding a multiple epitope fusion polypeptide sequence according to any preceding claim or a complement thereof.
  6. A recombinant vector comprising: (a) a polynucleotide according to Claim 5; and (b) at least one control element operably linked to the polynucleotide, whereby the coding sequence can be transcribed and translated in a host cell.
  7. A host cell comprising a recombinant vector according to Claim 6.
  8. A method for producing a multiple epitope fusion polypeptide, the method comprising culturing a cell according to Claim 7 under conditions for producing the polypeptide.
  9. A vaccine composition comprising a pharmaceutically acceptable vehicle and a multiple epitope fusion polypeptide according to any of Claims 1 to 4.
  10. A vaccine composition according to Claim 9, further comprising an adjuvant.
  11. A method of producing a vaccine composition comprising the steps of: (a) providing a multiple epitope fusion polypeptide according to any of Claims 1 to 4; and (b) combining the polypeptide with a pharmaceutically acceptable vehicle.
  12. A antibody directed against a multiple epitope fusion polypeptide according to any of Claims 1 to 4.
  13. An antibody according to Claim 12, in which the antibody is a polyclonal antibody.
  14. An antibody according to Claim 12, in which the antibody is a monoclonal antibody.
  15. An immunodiagnostic test kit for detecting Streptococcus infection, the test kit comprising a multiple epitope fusion polypeptide according to any of Claims 1 to 4, and instructions for conducting the immunodiagnostic test.
  16. Use of a multiple epitope fusion polypeptide according to any of Claims 1 to 4 for the manufacture of a vaccine composition useful for treating or preventing a bacterial infection in a vertebrate subject.
  17. Use according to Claim 16, in which the bacterial infection is a streptococcal infection.
  18. Use according to Claim 17, in which the bacterial infection causes mastitis.
  19. A method of detecting Streptococcus antibodies in a biological sample comprising: (a) reacting the biological sample with a multiple epitope fusion polypeptide according to any of Claims 1 to 4, under conditions which allow the Streptococcus antibodies, when present in the biological sample, to bind to the sequence to form an antibody/antigen complex; and (b) detecting the presence or absence of the complex, and thereby detecting the presence or absence of Streptococcus antibodies in the sample.
EP01944803A 2000-06-12 2001-06-11 Chimeric GapC protein from Streptococcus and its use in vaccination and diagnosis Expired - Lifetime EP1294771B1 (en)

Priority Applications (1)

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CY20091100094T CY1108724T1 (en) 2000-06-12 2009-01-27 IMMUNIZATION OF CAT MILK PRODUCTS WITH CHEMICAL GAPC PROTEIN AGAINST streptococcal infection

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PT1294771E (en) 2009-01-06
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US20050142615A1 (en) 2005-06-30
DK1294771T3 (en) 2008-12-15
US6875853B2 (en) 2005-04-05
WO2001096379A3 (en) 2002-08-08
AR028713A1 (en) 2003-05-21
US7258992B2 (en) 2007-08-21
DE60136356D1 (en) 2008-12-11
WO2001096379A2 (en) 2001-12-20
ES2316450T3 (en) 2009-04-16
ATE412673T1 (en) 2008-11-15
US20040062774A1 (en) 2004-04-01
US20020044928A1 (en) 2002-04-18
CA2411919C (en) 2012-07-31
US20080009022A1 (en) 2008-01-10
US6660270B2 (en) 2003-12-09
CY1108724T1 (en) 2014-04-09

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